Transthyretin antibodies and uses thereof

ABSTRACT

The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to human transthyretin (TTR). The anti-TTR antibodies or antigen-binding fragments thereof are useful, for example, in detecting TTR and in treating TTR amyloidosis in a subject.

BACKGROUND OF THE INVENTION Field of the Invention

The present disclosure provides antibodies and antigen-binding fragmentsthereof that bind to transthyretin (TTR) and methods of using the same.

Background

Transthyretin (TTR), also termed prealbumin, is a soluble, β-sheet rich,127-amino acid, non-glycosylated protein primarily synthesized andsecreted into the blood by the liver. TTR circulates in the bloodpredominantly as a 55 kDa tetramer, with a minor concentration ofdissociated monomer.

The monomeric subunits of TTR fold into a β-sandwich tertiary structurethat spontaneously assembles into a tetramer within the cellularendoplasmic reticulum. The tetramer exhibits two distinct dimer-dimerinterfaces, the less stable of which makes up two highly conservedthyroxine (T4) binding sites. The three-dimensional structures of theTTR tetramer bound to T4 and its other known ligand, the vitaminA-retinol-binding protein complex, have been solved.

Serum levels of TTR appear to mirror total body nitrogen and total bodypotassium levels such that they have been suggested to be a marker ofnutrition and inflammation, apparently serving as a negative acute phasereactant. The half-life of TTR in blood in vivo is twenty-four toforty-eight hours.

In blood, the TTR tetramer binds retinol-binding protein bound toretinol (holo-retinol-binding protein) and a small amount of T4, andtransports them to tissues on the minute-to-hour timescale. Albumin hasthe highest plasma concentration carrying capacity for T4, and thyroidbinding globulin has the highest affinity for T4. Thus, TTR apparentlyplays a minor role in the physiological transport of T4.

TTR-Triggered Amyloidogenesis

In vitro studies suggest that tetrameric TTR does not form amyloidfibrils and that amyloidogenesis follows tetramer dissociation intodimers, which then rapidly dissociate to folded monomers. Amyloidfibrillogenesis results from exposure of stretches of hydrophobicresidues in TTR monomers that subsequently misassemble into sphericalaggregates, which then undergo conformational conversion into cross-0sheet assemblies. Tetramer dissociation is thought to be rate limitingfor TTR amyloid formation in the case of the wild-type (WT) protein, andmost tetramers produced by heterozygotes contain both mutant and WT TTRsubunits.

Clinical Overview of TTR Amyloidosis

The hereditary TTR amyloidosis are rare, autosomal dominant diseasescaused by point mutations in the TTR gene. More than one hundreddestabilizing mutations of TTR have been identified.

Although the main pathologic manifestation of hereditary TTR amyloidosisis a progressive ascending polyneuropathy and/or cardiomyopathy, mixedphenotypes, extreme variability in the clinical presentation, andincomplete penetrance often result in misdiagnosis by physicians notconsidering TTR amyloidosis.

Most known cases of TTR-Familial Amyloidotic Polyneuropathy (FAP) resultfrom the Va130Met (p.Va150Met) mutation, frequent in regions ofPortugal, Japan, Sweden, and Brazil. TTR-FAP is characterized by asensory, motor, and autonomic neuropathy. Gastrointestinal symptoms area common complication of autonomic neuropathy. Amyloid deposits,typically found in ganglia, the endoneurium, and nerve blood vesselgradually lead to destruction of unmyelinated nerve fibers, and thensmall and large myelinated nerve fibers. Distal axonal degeneration isthe main pathogenic feature. Potential underlying mechanisms include:(i) direct toxicity and mechanical stress imposed by modified TTRdeposits on nerve fibers; and/or (ii) toxicity of non-amyloid TTR.

Clinical manifestations of cardiac involvement may follow later in thecourse of disease, but, silent cardiovascular features suggesting heartinvolvement are not uncommon. Neurologic and cardiac deteriorationprogress gradually during amyloidogenesis, typically leading to deathwithin five to fifteen years of symptom onset.

In the predominant cardiac phenotype, namely, TTR amyloidcardiomyopathy, amyloid infiltrates unselectively, the valves,ventricles, atria, and the conduction system, leading to leftventricular wall thickening with a non-dilated left ventricle andimpaired longitudinal contraction, and results in congestive heartfailure and arrhythmias. Mean survival, although not adequatelydocumented is in the range of two years from diagnosis, with deathprimarily from heart failure or sudden death.

The most prevalent mutation associated with TTR-Familial AmyloidoticCardiomyopathy (FAC) is Va1122Ile (p.Va1142Ile), which is found in up to4% of African Americans. Other pathogenic TTR mutations are less commonresulting in a “mixed” phenotype of neurologic and multi-organ systeminvolvement.

In wild-type (WT) TTR amyloidosis, WT TTR tetramers dissociate intomonomers, misfold, and undergo amyloidogenesis. Post mortem studiessuggest TTR wt phenotype in up to 22-25% of individuals over 80 years ofage, and 32% of adults over the age of 75 with heart failure and apreserved ejection fraction. WT TTR amyloidosis is usually a late-onset,sporadic condition primarily affecting men. The prevalence of nervoussystem involvement among patients with symptomatic WT TTR amyloidosis isapproximately 28%, and nearly half have a prior carpal tunnel syndromeapparently owing to amyloid deposition in the connective tissue of thetransverse carpal ligament.

Treatment

The only approved treatment for familial TTR amyloidosis is orthotopicliver transplantation. However, WT TTR patients are generally older andunable to tolerate organ transplantation. Additionally, cardiac andperipheral nerve deposition of amyloid continue to progress, despiteliver transplant.

Several TTR-stabilizing agents have been developed and are in variousstages of clinical trials. Diflunisal, a nonsteroidal anti-inflammatorydrug (NSAID), binds and stabilizes common familial TTR variants againstacid-mediated fibril formation in vitro and has been tested in humanclinical trials. Use of diflunisal in transthyretin cardiac amyloidosis(ATTR-CA) is controversial given known consequences of chronicinhibition of cyclooxygenase (COX) enzymes, including gastrointestinalbleeding (COX-1), renal dysfunction (COX-2), fluid retention, andhypertension that may precipitate heart failure (COX-2) in vulnerableindividuals.

Tafamidismeglumine (“tafamidis”) is a benzoxazole lacking NSAIDactivity, which makes it ideal for heart failure patients for whom thefluid retention and renal dysfunction caused by NSAIDs may worsendisease manifestations. Tafamid is kinetically stabilizes TTR andinhibits amyloidogenesis. In a double-blind, placebo-controlled,randomized trial of patients with FAP, tafamidis appeared to reduceperipheral neurologic impairment, though the co-primary endpoints, basedon the intention to treat two different neuropathy symptom-scoringsystems, were not significantly different between treatment and placeboarms. Tafamidis has been approved in Europe and other locations forearly stage FAP, but it is still under review in the United States. Moreclinical trials are underway to evaluate its efficacy in TTR cardiacamyloid patients.

Degradation of TTR mRNAs either by short interfering RNA (siRNA) orantisense oligonucleotides has been shown to be an effective method inlowering TTR serum level and halting the progression of amyloidformation. Ionis Pharmaceuticals and Alnylam Pharmaceuticals have bothrecently reported successful results of antisense oligonucleotides andsiRNA, respectively, in FAP. Submissions to the FDA are ongoing.

Given the poor prognosis for patients with TTR amyloidosis, superiordetection and treatment reagents are necessary.

BRIEF SUMMARY OF THE INVENTION

Provided herein are antibodies and antigen-binding fragments thereofcapable of binding human transthyretin (TTR) and methods of using thesame.

In one aspect, an antibody or antigen-binding fragment thereof capableof binding TTR, comprises a complementary determining region (CDR) H1comprising the amino acid sequence set forth in SEQ ID NO:1 (GYTFTSYY),a CDR H2 comprising the amino acid sequence set forth in SEQ ID NO:2(IYPGNVNT), a CDR H3 comprising the amino acid sequence set forth in SEQID NO:3 (ARTYFDY), a CDR L1 comprising the amino acid sequence set forthin SEQ ID NO:4 (SSVSY), a CDR L2 comprising the amino acid sequence setforth in SEQ ID NO:5 (DTS), and a CDR L3 comprising the amino acidsequence set forth in SEQ ID NO:6 (QQWSSKSFT).

In one aspect, an antibody or antigen-binding fragment thereof capableof binding TTR, comprises a complementary determining region (CDR) H1comprising the amino acid sequence set forth in SEQ ID NO:8 (SYYIH), aCDR H2 comprising the amino acid sequence set forth in SEQ ID NO:9(WIYPGNVNTKYNEKFKG), a CDR H3 comprising the amino acid sequence setforth in SEQ ID NO:10 (TYFDY), a CDR L1 comprising the amino acidsequence set forth in SEQ ID NO:11 (SASSSVSYMH), a CDR L2 comprising theamino acid sequence set forth in SEQ ID NO:12 (DTSKLAS), and a CDR L3comprising the amino acid sequence set forth in SEQ ID NO:6 (QQWSSKSFT).

In one aspect, the antibody or antigen-binding fragment comprises aheavy chain variable region (VH) and a light chain variable region (VL),wherein the VH comprises the same amino acid sequence as the VH of theantibody produced by the hybridoma cell line deposited at the ATCC® asdeposit number PTA-125005 on Mar. 14, 2018.

In one aspect, the antibody or antigen-binding fragment comprises aheavy chain variable region (VH) and a light chain variable region (VL),wherein the VL comprises the same amino acid sequence as the VL of theantibody produced by the hybridoma of ATCC® deposit number PTA-125005.

In one aspect, an antibody or antigen-binding fragment thereof thatspecifically binds to human TTR comprises a VH and a VL, wherein the VHcomprises the same amino acid sequence as the VH of the antibodyproduced by the hybridoma of ATCC® deposit number PTA-125005.

In one aspect, an antibody or antigen-binding fragment thereof thatspecifically binds to human TTR comprises a VH and a VL, wherein the VLcomprises the same amino acid sequence as the VL of the antibodyproduced by the hybridoma of ATCC® deposit number PTA-125005.

In one aspect, an antibody or antigen-binding fragment thereof capableof binding human TTR comprises a VH comprising the same amino acidsequence as the VH of the antibody produced by the hybridoma of ATCC®deposit number PTA-125005 and a VL comprising the same amino acidsequence as the VL of the antibody produced by the hybridoma of ATCC®deposit number PTA-125005.

In one aspect, the antibody or antigen-binding fragment furthercomprises a heavy chain constant region. In one aspect, the heavy chainconstant region is a human IgG₁ heavy chain constant region.

In one aspect, the antibody or antigen-binding fragment furthercomprises a light chain constant region. In one aspect, the light chainconstant region is a human IgGκ light chain constant region

In one aspect, the antibody or antigen-binding fragment thereofcomprises a heavy chain comprising the same amino acid sequence as theheavy chain of the antibody produced by the hybridoma of ATCC® depositnumber PTA-125005.

In one aspect, the antibody or antigen-binding fragment thereofcomprises a light chain comprising the same amino acid sequence as thelight chain of the antibody produced by the hybridoma of ATCC® depositnumber PTA-125005.

In one aspect, the antibody or antigen-binding fragment comprises aheavy chain and a light chain comprising the same amino acid sequence asthe heavy chain of the antibody produced by the hybridoma of ATCC®deposit number PTA-125005 and the same amino acid sequence as the lightchain of the antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, respectively.

In one aspect, an antibody or antigen-binding fragment thereof thatspecifically binds to human TTR comprises the CDR H1, CDR H2, CDR H3,CDR L1, CDR L2, and CDR L3 of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005.

In one aspect, the CDRs are the Kabat-defined CDRs, the Chothia-definedCDRs, or the AbM-defined CDRs.

In one aspect, the antibody or antigen-binding fragment thereof binds tohuman TTR monomers. In one aspect, the antibody or antigen-bindingfragment thereof binds to human TTR fibrils.

In one aspect, the antibody or antigen-binding fragment thereof iscapable of decreasing the toxicity of TTR fibrils on humancardiomyocytes. In one aspect, the antibody or antigen-binding fragmentthereof is capable of inhibiting the accumulation of TTR aggregates inan organ. In one aspect, the organ is the heart and/or kidney.

Also provided herein are isolated antibodies or antigen-bindingfragments thereof that bind to the same epitope of human TTR as anyantibody or antigen-binding fragment thereof provided herein.

Also provided herein are isolated antibodies or antigen-bindingfragments thereof that competitively inhibit the binding of any antibodyor antigen-binding fragment thereof provided herein to human TTR.

In one aspect, the antibody or antigen-binding fragment thereof is ahuman antibody or antigen-binding fragment thereof. In one aspect, theantibody or antigen-binding fragment thereof is a full-length antibody.In one aspect, the antibody or antigen-binding fragment thereof is anantigen-binding fragment. In one aspect, the antigen-binding fragmentcomprises a Fab, Fab′, F(ab′)₂, single chain Fv(scFv), disulfide linkedFv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab′)₃,tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab,mAb², (scFv)₂, or scFv-Fc.

In one aspect, the antibody or antigen-binding fragment thereof isisolated.

In one aspect, the antibody or antigen-binding fragment furthercomprises a detectable label.

Also provided herein are isolated polynucleotides comprising a nucleicacid molecule encoding the heavy chain variable region or heavy chain ofany antibody or antigen-binding fragment provided herein. In one aspect,the nucleic acid molecule encodes a VH comprising the same amino acidsequence as the VH of the antibody produced by the hybridoma of ATCC®deposit number PTA-125005. In one aspect, the nucleic acid moleculecomprises (i) the VH-encoding sequence in the hybridoma of ATCC® depositnumber PTA-125005 or (ii) the heavy chain-encoding sequence in thehybridoma of ATCC® deposit number PTA-125005.

Also provided herein are isolated polynucleotides comprising a nucleicacid molecule encoding the light chain variable region or light chain ofany antibody or antigen-binding fragment provided herein. In one aspect,the nucleic acid molecule encodes a VL comprising the same amino acidsequence as the VL of the antibody produced by the hybridoma of ATCC® asdeposit number PTA-125005. In one aspect, the nucleic acid moleculecomprises (i) the VL-encoding sequence in the hybridoma of ATCC® depositnumber PTA-125005, (ii) the light chain-encoding sequence in thehybridoma of ATCC® deposit number PTA-125005.

Also provided herein are isolated polynucleotides comprising a nucleicacid molecule encoding the heavy chain variable region or heavy chain ofany antibody or antigen-binding fragment thereof provided herein and thelight chain variable region or light chain of any antibody orantigen-binding fragment thereof provided herein.

Also provided herein are isolated vectors comprising any polynucleotideprovided herein.

Also provided herein are host cells comprising any polynucleotideprovided herein, any vector provided herein, or any combination ofvectors comprising polynucleotides provided herein. In one aspect, thehost cell is selected from the group consisting of CHO, HEK-293T, HeLaand BHK cells, optionally wherein the CHO cell is a CHO-K1SP cell.

Also provided herein are methods of producing an antibody orantigen-binding fragment thereof that binds to human TTR comprisingculturing any host cell provided herein so that the nucleic acidmolecule is expressed and the antibody or antigen-binding fragmentthereof is produced.

Also provided herein are isolated antibodies or antigen-bindingfragments thereof that are produced by any method provided herein.

Also provided herein are pharmaceutical compositions comprising anyantibody or antigen-binding fragment thereof provided herein and apharmaceutically acceptable excipient.

Also provided herein are methods for detecting TTR in a samplecomprising contacting the sample with any antibody or antigen-bindingfragment thereof provided herein.

Also provided herein are methods of precipitating TTR from a samplecomprising contacting the sample with any antibody or antigen-bindingfragment thereof provided herein. In one aspect, the sample is obtainedfrom a subject suspected of having a TTR amyloidosis.

Also provided herein are methods for increasing the uptake of TTRaggregates by an immune cell exposed to TTR aggregates comprisingcontacting the immune cell with any antibody or antigen-binding fragmentprovided herein or any pharmaceutical composition provided herein. Inone aspect, the immune cell is a monocyte. In one aspect, the immunecell is a microglia.

Also provided herein are methods for decreasing the toxicity of TTRaggregates on a cell exposed to TTR aggregates comprising contacting thecell with any antibody or antigen-binding fragment thereof providedherein or any pharmaceutical composition provided herein. In one aspect,the cell is a cardiomyocyte.

Also provided herein are methods of inhibiting the accumulation of TTRaggregates in an organ exposed to TTR aggregates comprising contactingthe organ with any antibody or antigen-binding fragment thereof providedherein or any pharmaceutical composition provided herein. In one aspect,the organ is a heart. In one aspect, the organ is a kidney. In oneaspect, the contacting occurs in vitro. In one aspect, the contactingoccurs in a subject.

Also provided herein are methods of treating or preventing a TTRamyloidosis in a subject comprising administering to the subject anyantibody or antigen-binding fragment thereof provided herein or anypharmaceutical composition provided herein. In one aspect, the TTRamyloidosis is TTR-Familial Amyloidotic Polyneuropathy (FAP). In oneaspect, the TTR amyloidosis is TTR-Familial Amyloidotic Cardiomyopathy(FAC). In one aspect, the TTR amyloidosis is TTR amyloid cardiomyopathy.In one aspect, the TTR amyloidosis is wild-type (WT) TTR amyloidosis.

Also provided herein are methods for detecting TTR in a subjectcomprising administering to the subject any antibody or antigen-bindingfragment thereof provided herein or any pharmaceutical compositionprovided herein.

Also provided herein are methods for diagnosing a TTR amyloidosis in asubject comprising administering to the subject any antibody orantigen-binding fragment thereof provided herein or any pharmaceuticalcomposition provided herein. In one aspect, the antibody orantigen-binding fragment thereof is linked to a detection reagent,optionally wherein the detection reagent is biotin, horse radishperoxidase (HRP), Cy5, FITC/Cy3, Alexa-488, or alkaline phosphatase.

In one aspect, the subject is a human.

Also provided herein are kits comprising any antibody or antigen-bindingfragment thereof provided herein or any pharmaceutical compositionprovided herein and a) a detection reagent, b) TTR antigen, c) a noticethat reflects approval for use or sale for human administration, or d) acombination thereof.

Also provided herein are methods for testing the activity of aTTR-binding compound comprising administering the TTR-binding compoundand TTR fibrils to a rodent. In one aspect, the TTR fibrils areadministered by injecting the TTR fibrils into an organ of the rodent.In one aspect, the organ is a heart. In one aspect, the organ is akidney. In one aspect, the organ is a brain. In one aspect, theTTR-binding compound is administered after the TTR fibrils are injected.In one aspect, the TTR-binding compound is administered before the TTRfibrils are injected. In one aspect, the TTR-binding compound isadministered with the TTR fibrils. In one aspect, the TTR fibrils areadministered with a gelatinous protein mixture secreted byEngelbreth-Holm-Swarm (EHS) mouse sarcoma cells. In one aspect, themethods further comprise administering technetium pyrophosphate(^(99m)Tc-PYP) to the rodent after injection of the TTR fibrils. In oneaspect, the ^(99m)Tc-PYP is administered intravenously. In one aspect,the methods further comprise detecting the TTR fibrils and/or the^(99m)Tc-PYP. In one aspect, the TTR fibrils and/or ^(99m)Tc-PYP isdetected in the rodent. In one aspect, the TTR fibrils and/or^(99m)Tc-PYP is detected in an organ that has been removed from therodent. In one aspect, the ^(99m)Tc-PYP is detected using Computertomography (CT) scanning. In one aspect, the rodent is a rat. In oneaspect, the rat is a Sprague-Dawley rat.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1A is a graph showing binding of the mouse IgG antibodies Ab-A,Ab-B, CGX304, and Ab-C to transthyretin (TTR) in monomer and fibrillarforms. (See Example 1.)

FIG. 1B is a graph showing binding of the mouse IgG antibodies Ab-D,Ab-E, and Ab-F and a sham antibody to TTR in monomer and fibrillarforms. (See Example 1.)

FIG. 2 is a graph showing the binding of CGX304, Ab-D, and shame (1G4-2)antibodies to monomer and fibrillar TTR by ELISA (See Example 2.)

FIG. 3 is a table showing the binding of CGX304 to monomer and fibrillarTTR as measured by surface plasmon resonance (Biacore). “Ka” refers tothe associate rate constant; “kd” refers to the dissociate rateconstant; and “KD” refers to the affinity constant. (See Example 2.)

FIG. 4 shows immunoprecipitation of recombinant human TTR (“hTTR”) orTTR from TTR-amyloidosis patient sera using CGX304 or a negativecontrol. The TTR staining was performed using mouse monoclonalanti-human TTR antibody. (See Example 3.)

FIG. 5 shows immunohistochemical staining of a human heart tissue sampleobtained from a TTR-amyloidosis patient using CGX304 (right panels) andcongo red staining of the same human heart tissue sample (left panels).(See Example 4.)

FIG. 6 is a bar graph showing the effect of CGX304 and a controlantibody (1G4-2) on the viability of cardiomyocytes (H9c2 cell line)exposed to 2.5 μM fibrillar TTR. The y-axis represents the relativeincrease in cell viability resulting from treating with CGX304 or thecontrol antibody (1G4-2) as compared to treatment with PBS. (See Example5.)

FIG. 7A is a bar graph showing the uptake of fluorescent fibrillar TTRby mouse microglia BV2 cells. The y-axis represents the relativegeometric mean (gMFI) measured by flow cytometry. (See Example 6.)

FIG. 7B is a bar graph showing the uptake of fluorescent fibrillar TTRby human monocytic THP-1 cells. The y-axis represents the relativegeometric mean (gMFI) measured by flow cytometry. (See Example 6.)

FIG. 7C shows the uptake of fibrillar TTR by human monocytes (U937) asmeasured by western blot. (See Example 6.)

FIG. 7D is a bar graph showing the uptake of fibrillar TTR by humanmonocytes (U937) by western blot. The y-axis represents the volumeintensity (pixels) normalized to GAPDH analyzed using image Lab software(Bio-rad). (See Example 6.)

FIG. 8A shows aggregated TTR detected in rodents by PYP scans combinedwith Computer CT. TTR was injected into the rodents' hearts. The rodentin the left panel was treated with sham, and the rodent in the rightpanel was treated with CGX304. The white arrows indicate aggregated TTRas seen in PET-CT images. (See Example 7.)

FIG. 8B is a bar graph showing data from image analysis of CT scansquantitating fluorescence intensity (F.I) levels in rodents thatreceived TTR injections in the heart and then treatment with eitherCGX304 or sham. The y-axis represents the F.I of the heart divided bythe F.I of the chest wall used as background. (See Example 7.)

FIG. 8C is a bar graph showing CGX304 levels in the heart of rats postCT experiment. Mouse IgG1 levels were quantified using ELISA detection.The y-axis represents the change in mouse IgG1 levels. (See Example 7.)

FIG. 9A shows aggregated TTR detected in a rodent by PET-CT. Therodent's left kidney was treated with CGX304, and the rodent's rightkidney was treated with sham. (See Example 7.)

FIG. 9B is a bar graph showing data from image analysis of CT scansquantitating relative F.I levels of left and right kidneys of three ratsthat were treated with CGX304 in the left kidney and sham in the rightkidney. The y-axis represents the F.I of the right kidney divided by theF.I of the left kidney. (See Example 7.)

FIG. 9C shows an image of a Western Blot showing TTR in CGX304-treatedkidney compared to sham-treated kidney. (See Example 7.)

FIG. 10A is a graph showing the serum concentration of CGX304 over 28days in animals that received intravenous (“IV”) injections of 0.5mg/kg, 2.5 mg/kg, or 10 mg/kg CGX304 (n=3, 3, 3). The y-axis representsthe CGX304 concentration levels. (See Example 8.)

FIG. 10B is a graph showing the serum concentration of TTR over time ina rat that received an IV injection of 2.5 mg/kg CGX304. The y-axisrepresents the change in TTR levels. (See Example 8.)

FIG. 10C is a bar graph showing CGX304 levels in the heart of rats 28days post IV treatment. CGX304 levels were quantified using ELISAdetection assay. The y-axis represents the change in mouse IgG1 levels.(See Example 8.)

FIG. 11 is representative immunohistochemical staining images on ratsinjected with aggregated TTR (right panel) or control (sham animalsinjected with matrigel and no aggregated TTR—left panel) showinginterstitial infiltration of aggregated TTR in the sections taken frommid and lower apical regions of respective rat hearts. (See Example 11.)

FIG. 12 shows immunohistochemical staining of a human heart tissuesample obtained from a TTR-amyloidosis patient using CGX304 (middlepanel), isotype control IgG (right panel) and congo red staining (leftpanel) of the same human heart tissue sample. (See Example 9.)

FIG. 13 is a bar graph showing data from image analysis of CT SPECTscans quantitating PYP uptake in rodents that received TTR injections inthe heart and were then treated with either CGX304 or sham. The y-axisrepresents the left ventricle uptake divided by the chest wall uptakeused as background. Error bars indicate SEM. (See Example 10.)

FIGS. 14A-14E show deformation indices measured before and afterinjection of TTR with and without CGX304. FIG. 14A shows the radialstrain change in the two groups. FIG. 14B shows the circumferentialstrain rate S change in the two groups. FIG. 14C shows the ratiocircumferential strain rate E to A in the two groups. FIG. 14D shows themyocardial radial strain before and after TTR injection with and withoutCGX304 treatment. The upper panel shows a decrease in Radial strainafter injection of TTR. The lower panel shows no significant change inradial strain after injection of TTR and CGX304. FIG. 14E showsBand-Altman analysis of intra-observer variability in RS and CSmeasurements. (See Example 12.)

FIG. 15 shows the results of ELISA assay testing in the presence ofcirculating aggregated TTR employing CGX304 in the sera of patients withwild-type (wt) ATTR cardiac amyloidosis. Samples were measured intriplicate. (See Example 13.)

DETAILED DESCRIPTION OF THE INVENTION I. Terminology

As used herein, the terms “transthyretin” and “TTR” are interchangeableand refer to mammalian TTR polypeptides including, but not limited to,wild-type and mutant TTR polypeptides. “TTR” encompasses full-length,unprocessed TTR polypeptides as well as forms of TTR polypeptides thatresult from processing within the cell. “TTR” encompasses polypeptidesin monomeric, tetrameric, and fibrillar forms. As used herein, the term“human TTR” refers to a polypeptide comprising amino acids 21-147 of SEQID NO:7. A “TTR polynucleotide,” “TTR nucleotide,” or “TTR nucleic acid”refer to a polynucleotide encoding TTR.

The term “antibody” means an immunoglobulin molecule that recognizes andspecifically binds to a target, such as a protein, polypeptide, peptide,carbohydrate, polynucleotide, lipid, or combinations of the foregoingthrough at least one antigen recognition site within the variable regionof the immunoglobulin molecule. As used herein, the term “antibody”encompasses intact polyclonal antibodies, intact monoclonal antibodies,chimeric antibodies, humanized antibodies, human antibodies, fusionproteins comprising an antibody, and any other modified immunoglobulinmolecule so long as the antibodies exhibit the desired biologicalactivity. An antibody can be of any the five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes)thereof (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on theidentity of their heavy-chain constant domains referred to as alpha,delta, epsilon, gamma, and mu, respectively. The different classes ofimmunoglobulins have different and well known subunit structures andthree-dimensional configurations. Antibodies can be naked or conjugatedto other molecules such as toxins, radioisotopes, etc.

The term “antibody fragment” refers to a portion of an intact antibody.An “antigen-binding fragment,” “antigen-binding domain,” or“antigen-binding region,” refers to a portion of an intact antibody thatbinds to an antigen. An antigen-binding fragment can contain an antigenrecognition site of an intact antibody (e.g., complementaritydetermining regions (CDRs) sufficient to bind antigen). Examples ofantigen-binding fragments of antibodies include, but are not limited toFab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, and singlechain antibodies. An antigen-binding fragment of an antibody can bederived from any animal species, such as rodents (e.g., mouse, rat, orhamster) and humans or can be artificially produced.

The terms “anti-TTR antibody,” “TTR antibody,” and “antibody that bindsto TTR” refer to an antibody that is capable of binding TTR (inmonomeric, tetrameric, and/or fibrillar form) with sufficient affinitysuch that the antibody is useful as a diagnostic and/or therapeuticagent in targeting TTR. The extent of binding of an anti-TTR antibody toan unrelated, non-TTR protein can be less than about 10% of the bindingof the antibody to TTR as measured, e.g., by a radioimmunoassay (MA).

A “monoclonal” antibody or antigen-binding fragment thereof refers to ahomogeneous antibody or antigen-binding fragment population involved inthe highly specific recognition and binding of a single antigenicdeterminant, or epitope. This is in contrast to polyclonal antibodiesthat typically include different antibodies directed against differentantigenic determinants. The term “monoclonal” antibody orantigen-binding fragment thereof encompasses both intact and full-lengthmonoclonal antibodies as well as antibody fragments (such as Fab, Fab′,F(ab′)2, Fv), single chain (scFv) mutants, fusion proteins comprising anantibody portion, and any other modified immunoglobulin moleculecomprising an antigen recognition site. Furthermore, “monoclonal”antibodies or antigen-binding fragments thereof refers to suchantibodies and antigen-binding fragments thereof made in any number ofmanners including but not limited to by hybridoma, phage selection,recombinant expression, and transgenic animals.

As used herein, the terms “variable region” or “variable domain” areused interchangeably and are common in the art. The variable regiontypically refers to a portion of an antibody, generally, a portion of alight or heavy chain, typically about the amino-terminal 110 to 120amino acids or 110 to 125 amino acids in the mature heavy chain andabout 90 to 115 amino acids in the mature light chain, which differ insequence among antibodies and are used in the binding and specificity ofa particular antibody for its particular antigen. The variability insequence is concentrated in those regions called complementaritydetermining regions (CDRs) while the more highly conserved regions inthe variable domain are called framework regions (FR). Without wishingto be bound by any particular mechanism or theory, it is believed thatCDRs of the light and heavy chains are primarily responsible for theinteraction and specificity of the antibody with antigen. In certainaspects, the variable region is a human variable region. In certainaspects, the variable region comprises rodent or murine CDRs and humanframework regions (FRs). In particular aspects, the variable region is aprimate (e.g., non-human primate) variable region. In certain aspects,the variable region comprises rodent or murine CDRs and primate (e.g.,non-human primate) framework regions (FRs).

The terms “VL” and “VL domain” are used interchangeably to refer to thelight chain variable region of an antibody.

The terms “VH” and “VH domain” are used interchangeably to refer to theheavy chain variable region of an antibody.

The term “Kabat numbering” and like terms are recognized in the art andrefer to a system of numbering amino acid residues in the heavy andlight chain variable regions of an antibody or an antigen-bindingfragment thereof. In certain aspects, CDRs can be determined accordingto the Kabat numbering system (see, e.g., Kabat E A & Wu T T (1971) AnnNY Acad Sci 190: 382-391 and Kabat E A et al., (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242). Using the Kabatnumbering system, CDRs within an antibody heavy chain molecule aretypically present at amino acid positions 31 to 35, which optionally caninclude one or two additional amino acids, following 35 (referred to inthe Kabat numbering scheme as 35A and 35B) (CDR H1), amino acidpositions 50 to 65 (CDR H2), and amino acid positions 95 to 102 (CDRH3). Using the Kabat numbering system, CDRs within an antibody lightchain molecule are typically present at amino acid positions 24 to 34(CDR L1), amino acid positions 50 to 56 (CDR L2), and amino acidpositions 89 to 97 (CDR L3). In a specific aspect, the CDRs of theantibodies described herein have been determined according to the Kabatnumbering scheme.

Chothia refers instead to the location of the structural loops (Chothiaand Lesk, J. Mol. Biol. 196:901-917 (1987)). The end of the ChothiaCDR-H1 loop when numbered using the Kabat numbering convention variesbetween H32 and H34 depending on the length of the loop (this is becausethe Kabat numbering scheme places the insertions at H35A and H35B; ifneither 35A nor 35B is present, the loop ends at 32; if only 35A ispresent, the loop ends at 33; if both 35A and 35B are present, the loopends at 34). The AbM hypervariable regions represent a compromisebetween the Kabat CDRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software.

Loop Kabat AbM Chothia L1 L24-L34 L24-L34 L24-L34 L2 L50-L56 L50-L56L50-L56 L3 L89-L97 L89-L97 L89-L97 H1 H31-H35B H26-H35B H26-H32 . . . 34(Kabat Numbering) H1 H31-H35 H26-H35 H26-H32 (Chothia Numbering) H2H50-H65 H50-H58 H52-H56 H3 H95-H102 H95-H102 H95-H102

As used herein, the terms “constant region” and “constant domain” areinterchangeable and have their meaning common in the art. The constantregion is an antibody portion, e.g., a carboxyl terminal portion of alight and/or heavy chain which is not directly involved in binding of anantibody to antigen but which can exhibit various effector functions,such as interaction with the Fc receptor. The constant region of animmunoglobulin molecule generally has a more conserved amino acidsequence relative to an immunoglobulin variable domain. In certainaspects, an antibody or antigen-binding fragment comprises a constantregion or portion thereof that is sufficient for antibody-dependentcell-mediated cytotoxicity (ADCC).

As used herein, the term “heavy chain” when used in reference to anantibody can refer to any distinct type, e.g., alpha (α), delta (δ),epsilon (ε), gamma (γ), and mu (μ), based on the amino acid sequence ofthe constant domain, which give rise to IgA, IgD, IgE, IgG, and IgMclasses of antibodies, respectively, including subclasses of IgG, e.g.,IgG₁, IgG₂, IgG₃, and IgG₄. Heavy chain amino acid sequences are wellknown in the art. In specific aspects, the heavy chain is a human heavychain.

As used herein, the term “light chain” when used in reference to anantibody can refer to any distinct type, e.g., kappa (κ) or lambda (λ)based on the amino acid sequence of the constant domains. Light chainamino acid sequences are well known in the art. In specific aspects, thelight chain is a human light chain.

The term “chimeric” antibodies or antigen-binding fragments thereofrefers to antibodies or antigen-binding fragments thereof wherein theamino acid sequence is derived from two or more species. Typically, thevariable region of both light and heavy chains corresponds to thevariable region of antibodies or antigen-binding fragments thereofderived from one species of mammals (e.g. mouse, rat, rabbit, etc.) withthe desired specificity, affinity, and capability while the constantregions are homologous to the sequences in antibodies or antigen-bindingfragments thereof derived from another (usually human) to avoideliciting an immune response in that species.

The term “humanized” antibody or antigen-binding fragment thereof refersto forms of non-human (e.g. murine) antibodies or antigen-bindingfragments that are specific immunoglobulin chains, chimericimmunoglobulins, or fragments thereof that contain minimal non-human(e.g., murine) sequences. Typically, humanized antibodies orantigen-binding fragments thereof are human immunoglobulins in whichresidues from the complementary determining region (CDR) are replaced byresidues from the CDR of a non-human species (e.g. mouse, rat, rabbit,hamster) that have the desired specificity, affinity, and capability(“CDR grafted”) (Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536(1988)). In some instances, the Fv framework region (FR) residues of ahuman immunoglobulin are replaced with the corresponding residues in anantibody or fragment from a non-human species that has the desiredspecificity, affinity, and capability. The humanized antibody orantigen-binding fragment thereof can be further modified by thesubstitution of additional residues either in the Fv framework regionand/or within the non-human CDR residues to refine and optimize antibodyor antigen-binding fragment thereof specificity, affinity, and/orcapability. In general, the humanized antibody or antigen-bindingfragment thereof will comprise substantially all of at least one, andtypically two or three, variable domains containing all or substantiallyall of the CDR regions that correspond to the non-human immunoglobulinwhereas all or substantially all of the FR regions are those of a humanimmunoglobulin consensus sequence. The humanized antibody orantigen-binding fragment thereof can also comprise at least a portion ofan immunoglobulin constant region or domain (Fc), typically that of ahuman immunoglobulin. Examples of methods used to generate humanizedantibodies are described in U.S. Pat. No. 5,225,539; Roguska et al.,Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al.,Protein Eng. 9(10):895-904 (1996). In some aspects, a “humanizedantibody” is a resurfaced antibody.

The term “human” antibody or antigen-binding fragment thereof means anantibody or antigen-binding fragment thereof having an amino acidsequence derived from a human immunoglobulin gene locus, where suchantibody or antigen-binding fragment is made using any technique knownin the art. This definition of a human antibody or antigen-bindingfragment thereof includes intact or full-length antibodies and fragmentsthereof.

“Binding affinity” generally refers to the strength of the sum total ofnon-covalent interactions between a single binding site of a molecule(e.g., an antibody or antigen-binding fragment thereof) and its bindingpartner (e.g., an antigen). Unless indicated otherwise, as used herein,“binding affinity” refers to intrinsic binding affinity which reflects a1:1 interaction between members of a binding pair (e.g., antibody orantigen-binding fragment thereof and antigen). The affinity of amolecule X for its partner Y can generally be represented by thedissociation constant (K_(D)). Affinity can be measured and/or expressedin a number of ways known in the art, including, but not limited to,equilibrium dissociation constant (K_(D)), and equilibrium associationconstant (K_(A)). The K_(D) is calculated from the quotient ofk_(off)/k_(on), whereas K_(A) is calculated from the quotient ofk_(on)/k_(off). k_(on) refers to the association rate constant of, e.g.,an antibody or antigen-binding fragment thereof to an antigen, andk_(off) refers to the dissociation of, e.g., an antibody orantigen-binding fragment thereof from an antigen. The k_(on) and k_(off)can be determined by techniques known to one of ordinary skill in theart, such as BIAcore® or KinExA.

As used herein, an “epitope” is a term in the art and refers to alocalized region of an antigen to which an antibody or antigen-bindingfragment thereof can specifically bind. An epitope can be, for example,contiguous amino acids of a polypeptide (linear or contiguous epitope)or an epitope can, for example, come together from two or morenon-contiguous regions of a polypeptide or polypeptides (conformational,non-linear, discontinuous, or non-contiguous epitope). In certainaspects, the epitope to which an antibody or antigen-binding fragmentthereof binds can be determined by, e.g., NMR spectroscopy, X-raydiffraction crystallography studies, ELISA assays, hydrogen/deuteriumexchange coupled with mass spectrometry (e.g., liquid chromatographyelectrospray mass spectrometry), array-based oligo-peptide scanningassays, and/or mutagenesis mapping (e.g., site-directed mutagenesismapping). For X-ray crystallography, crystallization may be accomplishedusing any of the known methods in the art (e.g., Giegé R et al., (1994)Acta Crystallogr D Biol Crystallogr 50(Pt 4): 339-350; McPherson A(1990) Eur J Biochem 189: 1-23; Chayen N E (1997) Structure 5:1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303).Antibody/antigen-binding fragment thereof: antigen crystals can bestudied using well known X-ray diffraction techniques and can be refinedusing computer software such as X-PLOR (Yale University, 1992,distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol(1985) volumes 114 & 115, eds Wyckoff H W et al.; U.S. 2004/0014194),and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed Carter C W;Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies can be accomplished using anymethod known to one of skill in the art. See, e.g., Champe M et al.,(1995) J Biol Chem 270: 1388-1394 and Cunningham B C & Wells J A (1989)Science 244: 1081-1085 for a description of mutagenesis techniques,including alanine scanning mutagenesis techniques.

A TTR antibody that “binds to the same epitope” as a reference TTRantibody refers to an antibody that binds to the same TTR amino acidresidues as the reference TTR antibody. The ability of a TTR antibody tobind to the same epitope as a reference TTR antibody is determined by ahydrogen/deuterium exchange assay (see Coales et al. Rapid Commun. MassSpectrom. 2009; 23: 639-647).

As used herein, the terms “immunospecifically binds,”“immunospecifically recognizes,” “specifically binds,” and “specificallyrecognizes” are analogous terms in the context of antibodies orantigen-binding fragments thereof. These terms indicate that theantibody or antigen-binding fragment thereof binds to an epitope via itsantigen-binding domain and that the binding entails some complementaritybetween the antigen-binding domain and the epitope. Accordingly, anantibody that “specifically binds” to human TTR (amino acids 21-147 ofSEQ ID NO:7) may also bind to TTR from other species (e.g., cynomolgusmonkey, mouse, and/or rat TTR) and/or TTR proteins produced from otherhuman alleles, but the extent of binding to an un-related, non-TTRprotein is less than about 10% of the binding of the antibody to TTR asmeasured, e.g., by a radioimmunoassay (MA).

An antibody is said to “competitively inhibit” binding of a referenceantibody to a given epitope if it preferentially binds to that epitopeor an overlapping epitope to the extent that it blocks, to some degree,binding of the reference antibody to the epitope. Competitive inhibitioncan be determined by any method known in the art, for example,competition ELISA assays. An antibody may be said to competitivelyinhibit binding of the reference antibody to a given epitope by at least90%, at least 80%, at least 70%, at least 60%, or at least 50%.

A polypeptide, antibody, polynucleotide, vector, cell, or compositionwhich is “isolated” is a polypeptide, antibody, polynucleotide, vector,cell, or composition which is in a form not found in nature. Isolatedpolypeptides, antibodies, polynucleotides, vectors, cell or compositionsinclude those which have been purified to a degree that they are nolonger in a form in which they are found in nature. In some aspects, anantibody, polynucleotide, vector, cell, or composition which is isolatedis substantially pure. As used herein, “substantially pure” refers tomaterial which is at least 50% pure (i.e., free from contaminants), atleast 90% pure, at least 95% pure, at least 98% pure, or at least 99%pure.

The terms “polypeptide,” “peptide,” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer can be linear or branched, it can comprise modifiedamino acids, and it can be interrupted by non-amino acids. The termsalso encompass an amino acid polymer that has been modified naturally orby intervention; for example, disulfide bond formation, glycosylation,lipidation, acetylation, phosphorylation, or any other manipulation ormodification, such as conjugation with a labeling component. Alsoincluded within the definition are, for example, polypeptides containingone or more analogs of an amino acid (including, for example, unnaturalamino acids, etc.), as well as other modifications known in the art. Itis understood that, because the polypeptides of this invention are basedupon antibodies, in certain aspects, the polypeptides can occur assingle chains or associated chains.

“Percent identity” refers to the extent of identity between twosequences (e.g., amino acid sequences or nucleic acid sequences).Percent identity can be determined by aligning two sequences,introducing gaps to maximize identity between the sequences. Alignmentscan be generated using programs known in the art. For purposes herein,alignment of nucleotide sequences can be performed with the blastnprogram set at default parameters, and alignment of amino acid sequencescan be performed with the blastp program set at default parameters (seeNational Center for Biotechnology Information (NCBI) on the worldwideweb, ncbi.nlm.nih.gov).

As used herein, the term “host cell” can be any type of cell, e.g., aprimary cell, a cell in culture, or a cell from a cell line. In specificaspects, the term “host cell” refers to a cell transfected with anucleic acid molecule and the progeny or potential progeny of such acell. Progeny of such a cell may not be identical to the parent celltransfected with the nucleic acid molecule, e.g., due to mutations orenvironmental influences that may occur in succeeding generations orintegration of the nucleic acid molecule into the host cell genome.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. The formulation can be sterile.

The terms “administer,” “administering,” “administration,” and the like,as used herein, refer to methods that may be used to enable delivery ofa drug, e.g., an anti-TTR antibody or antigen-binding fragment thereofto the desired site of biological action (e.g., intravenousadministration). Administration techniques that can be employed with theagents and methods described herein are found in e.g., Goodman andGilman, The Pharmacological Basis of Therapeutics, current edition,Pergamon; and Remington's, Pharmaceutical Sciences, current edition,Mack Publishing Co., Easton, Pa.

As used herein, the terms “subject” and “patient” are usedinterchangeably. The subject can be an animal. In some aspects, thesubject is a mammal such as a non-human animal (e.g., cow, pig, horse,cat, dog, rat, mouse, monkey or other primate, etc.). In some aspects,the subject is a human.

The term “therapeutically effective amount” refers to an amount of atherapeutic, e.g., an anti-TTR antibody or antigen-binding fragmentthereof, effective to treat a disease or disorder in a subject. In thecase of TTR amyloidosis, the therapeutically effective amount of thetherapeutic can reduce the number and/or size of amyloid fibrils,attenuate polyneuropathy, attenuate and/or improve symptoms in patientswith ATTR-related heart failure, and/or decrease cardiomyopathy. A“prophylactically effective amount” refers to an amount effective toachieve the desired prophylactic result.

Terms such as “treating,” “treatment,” “to treat,” “alleviating,” and“to alleviate” refer to therapeutic measures that cure, slow down,lessen symptoms of, and/or halt progression of a pathologic condition ordisorder. Thus, those in need of treatment include those alreadydiagnosed with or suspected of having the disorder. In certain aspects,a subject is successfully “treated” for a TTR amyloidosis according tothe methods of the present invention if the patient shows one or more ofthe following: reduce the number of amyloid fibrils, decreasepolyneuropathy, and/or decrease cardiomyopathy.

Prophylactic or preventative measures refer to measures that preventand/or slow the development of a targeted pathological condition ordisorder, e.g., a TTR amyloidosis. Thus, those in need of prophylacticor preventative measures include those prone to have the pathologicalcondition or disorder and those in whom the pathological condition ordisorder is to be prevented.

The term “TTR amyloidosis” refers to a condition characterized by thebuildup of abnormal deposits of amyloid (amyloidosis) in the body'sorgans and tissues (e.g., the nervous system, cardiac tissue, kidneysand/or other organs). TTR amyloidosis includes neuropathic TTRamyloidosis, leptomeningeal TTR amyloidosis, renal amyloidosis andcardiac TTR amyloidosis.

As used in the present disclosure and claims, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise.

It is understood that wherever aspects are described herein with thelanguage “comprising,” otherwise analogous aspects described in terms of“consisting of” and/or “consisting essentially of” are also provided. Inthis disclosure, “comprises,” “comprising,” “containing” and “having”and the like can have the meaning ascribed to them in U.S. patent lawand can mean “includes,” “including,” and the like; “consistingessentially of” or “consists essentially” likewise has the meaningascribed in U.S. patent law and the term is open-ended, allowing for thepresence of more than that which is recited so long as basic or novelcharacteristics of that which is recited are not changed by the presenceof more than that which is recited, but excludes prior art embodiments.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive. The term “and/or” as used in aphrase such as “A and/or B” herein is intended to include both “A andB,” “A or B,” “A,” and “B.” Likewise, the term “and/or” as used in aphrase such as “A, B, and/or C” is intended to encompass each of thefollowing embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C;A and C; A and B; B and C; A (alone); B (alone); and C (alone).

As used herein, the terms “about” and “approximately,” when used tomodify a numeric value or numeric range, indicate that deviations of 5%to 10% above and 5% to 10% below the value or range remain within theintended meaning of the recited value or range.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein

II. Anti-Transthyretin (TTR) Antibodies and Antigen-Binding FragmentsThereof

In a specific aspect, provided herein are antibodies (e.g., monoclonalantibodies) and antigen-binding fragments thereof which specificallybind to transthyretin (TTR) (e.g., human TTR). The amino acid sequencesfor human TTR are known in the art and are also provided herein as aminoacids 24-147 of SEQ ID NO:7.

Human TTR: (SEQ ID NO: 7)MASHRLLLLCLAGLVEVSEAGPTGTGESKCPLMVKVLDAVRGSPAINVAVHVFRKAADDTWEPFASGKTSESGELHGLTTEEEFVEGIYKVEIDTKSYWKALGISPFHEHAEVVFTANDSGPRRYTIAALLSPYSYSTTAVVTNPKE(The underlined amino acids (amino acids 1-20) of SEQ ID NO:7 are thesignal sequence of human TTR.)

In certain aspects, an antibody or antigen-binding fragment thereof foruse in the methods described herein binds to human TTR and comprises thesix CDRs of the CGX304 antibody listed as provided in Tables 1 and 2.

TABLE 1 VH CDR Amino Acid Sequences Anti- CDR H1 CDR H2 CDR H3 body(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) CGX304- GYTFTSYY (SEQ IDIYPGNVNT (SEQ ID NO: 2) ARTYFDY (SEQ ID IMGT NO: 1) NO: 3) definedCGX304- SYYIH (SEQ ID NO: 8) WIYPGNVNTKYNEKFKG TYFDY (SEQ ID NO: 10)Kabat (SEQ ID NO: 9) defined

TABLE 2 VL CDR Amino Acid Sequences CDR L1 CDR L2 CDR L3 Antibody(SEQ ID NO:) (SEQ ID NO:) (SEQ ID NO:) CGX304 - SSVSY (SEQ IDDTS (SEQ ID NO: 5) QQWSSKSFT (SEQ ID IMGT defined NO: 4) NO: 6) CGX304 -SASSSVSYMH DTSKLAS (SEQ ID QQWSSKSFT (SEQ ID Kabat defined(SEQ ID NO: 11) NO: 12) NO: 6)

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR and comprises a VH comprising thesame amino acid sequence as the VH of the antibody produced by thehybridoma cell line deposited at the American Type Culture Collection(ATCC®) at 10801 University Blvd Manassas, Va., 20110 USA as depositnumber PTA-125005 on Mar. 14, 2018, e.g., in combination with a VL.

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR and comprises a VL comprising thesame amino acid sequence as the VL of the antibody produced by thehybridoma of ATCC® depositPTA-125005, e.g., in combination with a VH.

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR and comprises a VH comprising thesame amino acid sequence as the VH of the antibody produced by thehybridoma of ATCC® deposit number PTA-125005, and a VL comprising thesame amino acid sequence as the VL of the antibody produced by thehybridoma of ATCC® deposit number PTA-125005.

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR, comprises a VH comprising asequence at least 80% identical to the VH of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, and a VL comprising asequence at least 80% identical to the VL of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005. In certain aspects, anantibody or antigen-binding fragment thereof described herein binds tohuman TTR, comprises a VH comprising a sequence at least 85% identicalto the VH of the antibody produced by the hybridoma of ATCC® depositnumber PTA-125005, and a VL comprising a sequence at least 85% identicalto the VL of the antibody produced by the hybridoma of ATCC® depositnumber PTA-125005.

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR, comprises a VH comprising asequence at least 90% identical to the VH of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, and a VL comprising asequence at least 90% identical to the VL of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005. In certain aspects, anantibody or antigen-binding fragment thereof described herein binds tohuman TTR, comprises a VH comprising a sequence at least 95% identicalto the VH of the antibody produced by the hybridoma of ATCC® depositnumber PTA-125005, and a VL comprising a sequence at least 95% identicalto the VL of the antibody produced by the hybridoma of ATCC® depositnumber PTA-125005.

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR, comprises a VH comprising asequence at least 96% identical to the VH of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, and a VL comprising asequence at least 96% identical to the VL of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005. In certain aspects, anantibody or antigen-binding fragment thereof described herein binds tohuman TTR, comprises a VH comprising a sequence at least 97% identicalto the VH of the antibody produced by the hybridoma of ATCC® depositnumber PTA-125005, and a VL comprising a sequence at least 97% identicalto the VL of the antibody produced by the hybridoma of ATCC® depositnumber PTA-125005. In certain aspects, an antibody or antigen-bindingfragment thereof described herein binds to human TTR, comprises a VHcomprising a sequence at least 98% identical to the VH of the antibodyproduced by the hybridoma of ATCC® deposit number PTA-125005, and a VLcomprising a sequence at least 98% identical to the VL of the antibodyproduced by the hybridoma of ATCC® deposit number PTA-125005. In certainaspects, an antibody or antigen-binding fragment thereof describedherein binds to human TTR, comprises a VH comprising a sequence at least99% identical to the VH of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005, and a VL comprising a sequence at least99% identical to the VL of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005.

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR, comprises a VH comprising asequence at least 80% identical to the VH of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, and a VL comprising asequence at least 80% identical to the VL of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, wherein the antibodyor antigen-binding fragment thereof decreases the toxicity of TTRfibrils (e.g., on human cardiomyocytes). In certain aspects, an antibodyor antigen-binding fragment thereof described herein binds to human TTR,comprises a VH comprising a sequence at least 85% identical to the VH ofthe antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, and a VL comprising a sequence at least 85% identical to theVL of the antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, wherein the antibody or antigen-binding fragment thereofdecreases the toxicity of TTR fibrils (e.g., on human cardiomyocytes).

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR, comprises a VH comprising asequence at least 90% identical to the VH of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, and a VL comprising asequence at least 90% identical to the VL of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, wherein the antibodyor antigen-binding fragment thereof decreases the toxicity of TTRfibrils (e.g., on human cardiomyocytes). In certain aspects, an antibodyor antigen-binding fragment thereof described herein binds to human TTR,comprises a VH comprising a sequence at least 95% identical to the VH ofthe antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, and a VL comprising a sequence at least 95% identical to theVL of the antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, wherein the antibody or antigen-binding fragment thereofdecreases the toxicity of TTR fibrils (e.g., on human cardiomyocytes).

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR, comprises a VH comprising asequence at least 96% identical to the VH of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, and a VL comprising asequence at least 96% identical to the VL of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, wherein the antibodyor antigen-binding fragment thereof decreases the toxicity of TTRfibrils (e.g., on human cardiomyocytes). In certain aspects, an antibodyor antigen-binding fragment thereof described herein binds to human TTR,comprises a VH comprising a sequence at least 97% identical to the VH ofthe antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, and a VL comprising a sequence at least 97% identical to theVL of the antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, wherein the antibody or antigen-binding fragment thereofdecreases the toxicity of TTR fibrils (e.g., on human cardiomyocytes).In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR, comprises a VH comprising asequence at least 98% identical to the VH of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, and a VL comprising asequence at least 98% identical to the VL of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, wherein the antibodyor antigen-binding fragment thereof decreases the toxicity of TTRfibrils (e.g., on human cardiomyocytes). In certain aspects, an antibodyor antigen-binding fragment thereof described herein binds to human TTR,comprises a VH comprising a sequence at least 99% identical to the VH ofthe antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, and a VL comprising a sequence at least 99% identical to theVL of the antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, wherein the antibody or antigen-binding fragment thereofdecreases the toxicity of TTR fibrils (e.g., on human cardiomyocytes).

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR, comprises a VH comprising asequence at least 80% identical to the VH of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, and a VL comprising asequence at least 80% identical to the VL of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, wherein the antibodyor antigen-binding fragment thereof inhibits accumulation of TTRaggregates (e.g., in an organ such as a heart and/or kidney). In certainaspects, an antibody or antigen-binding fragment thereof describedherein binds to human TTR, comprises a VH comprising a sequence at least85% identical to the VH of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005, and a VL comprising a sequence at least85% identical to the VL of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005, wherein the antibody or antigen-bindingfragment thereof inhibits accumulation of TTR aggregates (e.g., in anorgan such as a heart and/or kidney).

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR, comprises a VH comprising asequence at least 90% identical to the VH of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, and a VL comprising asequence at least 90% identical to the VL of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, wherein the antibodyor antigen-binding fragment thereof inhibits accumulation of TTRaggregates (e.g., in an organ such as heart and/or kidney). In certainaspects, an antibody or antigen-binding fragment thereof describedherein binds to human TTR, comprises a VH comprising a sequence at least95% identical to the VH of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005, and a VL comprising a sequence at least95% identical to the VL of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005, wherein the antibody or antigen-bindingfragment thereof inhibits accumulation of TTR aggregates (e.g., in anorgan such as a heart and/or kidney).

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR, comprises a VH comprising asequence at least 96% identical to the VH of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, and a VL comprising asequence at least 96% identical to the VL of the antibody produced bythe hybridoma of ATCC® deposit number PTA-125005, wherein the antibodyor antigen-binding fragment thereof inhibits accumulation of TTRaggregates (e.g., in an organ such as a heart and/or kidney). In certainaspects, an antibody or antigen-binding fragment thereof describedherein binds to human TTR, comprises a VH comprising a sequence at least97% identical to the VH of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005, and a VL comprising a sequence at least97% identical to the VL of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005, wherein the antibody or antigen-bindingfragment thereof inhibits accumulation of TTR aggregates (e.g., in anorgan such as a heart and/or kidney). In certain aspects, an antibody orantigen-binding fragment thereof described herein binds to human TTR,comprises a VH comprising a sequence at least 98% identical to the VH ofthe antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, and a VL comprising a sequence at least 98% identical to theVL of the antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, wherein the antibody or antigen-binding fragment thereofinhibits accumulation of TTR aggregates (e.g., in an organ such as aheart and/or kidney). In certain aspects, an antibody or antigen-bindingfragment thereof described herein binds to human TTR, comprises a VHcomprising a sequence at least 99% identical to the VH of the antibodyproduced by the hybridoma of ATCC® deposit number PTA-125005, and a VLcomprising a sequence at least 99% identical to the VL of the antibodyproduced by the hybridoma of ATCC® deposit number PTA-125005, whereinthe antibody or antigen-binding fragment thereof inhibits accumulationof TTR aggregates (e.g., in an organ such as a heart and/or kidney).

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein may be described by its VL domain alone or its VHdomain alone. See, for example, Rader C et al., (1998) PNAS 95:8910-8915, which is incorporated herein by reference in its entirety,describing the humanization of the mouse anti-αvβ3 antibody byidentifying a complementing light chain or heavy chain, respectively,from a human light chain or heavy chain library, resulting in humanizedantibody variants having affinities as high or higher than the affinityof the original antibody. See also Clackson T et al., (1991) Nature 352:624-628, which is incorporated herein by reference in its entirety,describing methods of producing antibodies that bind a specific antigenby using a specific VL domain (or VH domain) and screening a library forthe complementary variable domains. The screen produced 14 new partnersfor a specific VH domain and 13 new partners for a specific VL domain,which were strong binders, as determined by ELISA. See also Kim S J &Hong H J, (2007) J Microbiol 45: 572-577, which is incorporated hereinby reference in its entirety, describing methods of producing antibodiesthat bind a specific antigen by using a specific VH domain and screeninga library (e.g., human VL library) for complementary VL domains; theselected VL domains in turn could be used to guide selection ofadditional complementary (e.g., human) VH domains.

In certain aspects, the CDRs of an antibody or antigen-binding fragmentthereof can be determined according to the Chothia numbering scheme,which refers to the location of immunoglobulin structural loops (see,e.g., Chothia C & Lesk A M, (1987), J Mol Biol 196: 901-917; Al-LazikaniB et al., (1997) J Mol Biol 273: 927-948; Chothia C et al., (1992) J MolBiol 227: 799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226). Typically, when using the Kabatnumbering convention, the Chothia CDR-H1 loop is present at heavy chainamino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop is present atheavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop is presentat heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop ispresent at light chain amino acids 24 to 34, the Chothia CDR-L2 loop ispresent at light chain amino acids 50 to 56, and the Chothia CDR-L3 loopis present at light chain amino acids 89 to 97. The end of the ChothiaCDR-H1 loop when numbered using the Kabat numbering convention variesbetween H32 and H34 depending on the length of the loop (this is becausethe Kabat numbering scheme places the insertions at H35A and H35B; ifneither 35A nor 35B is present, the loop ends at 32; if only 35A ispresent, the loop ends at 33; if both 35A and 35B are present, the loopends at 34).

In certain aspects, provided herein are antibodies and antigen-bindingfragments thereof that specifically bind to TTR (e.g., human TTR) andcomprise the Chothia VH and VL CDRs of an antibody. In certain aspects,antibodies or antigen-binding fragments thereof that specifically bindto TTR (e.g., human TTR) comprise one or more CDRs, in which the Chothiaand Kabat CDRs have the same amino acid sequence. In certain aspects,provided herein are antibodies and antigen-binding fragments thereofthat specifically bind to TTR (e.g., human TTR) and comprisecombinations of Kabat CDRs and Chothia CDRs.

In certain aspects, the CDRs of an antibody or antigen-binding fragmentthereof can be determined according to the IMGT numbering system asdescribed in Lefranc M-P, (1999) The Immunologist 7: 132-136 and LefrancM-P et al., (1999) Nucleic Acids Res 27: 209-212. According to the IMGTnumbering scheme, CDR H1 is at positions 26 to 35, CDR H2 is atpositions 51 to 57, CDR H3 is at positions 93 to 102, CDR L1 is atpositions 27 to 32, CDR L2 is at positions 50 to 52, and CDR L3 is atpositions 89 to 97. In a particular aspect, provided herein areantibodies and antigen-binding fragments thereof that specifically bindto TTR (e.g., human TTR) and comprise the IMGT VH and VL CDRs of anantibody listed in Tables 3 and 4, for example, as described in LefrancM-P (1999) supra and Lefranc M-P et al., (1999) supra).

In certain aspects, the CDRs of an antibody or antigen-binding fragmentthereof can be determined according to MacCallum R M et al., (1996) JMol Biol 262: 732-745. See also, e.g., Martin A. “Protein Sequence andStructure Analysis of Antibody Variable Domains,” in AntibodyEngineering, Kontermann and Dübel, eds., Chapter 31, pp. 422-439,Springer-Verlag, Berlin (2001). In a particular aspect, provided hereinare antibodies or antigen-binding fragments thereof that specificallybind to TTR (e.g., human TTR) are determined by the method in MacCallumR M et al.

In certain aspects, the CDRs of an antibody or antigen-binding fragmentthereof can be determined according to the AbM numbering scheme, whichrefers AbM hypervariable regions which represent a compromise betweenthe Kabat CDRs and Chothia structural loops, and are used by OxfordMolecular's AbM antibody modeling software (Oxford Molecular Group,Inc.). In a particular aspect, provided herein are antibodies orantigen-binding fragments thereof that specifically bind to TTR (e.g.,human TTR) are determined by the AbM numbering scheme.

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR and comprises a heavy chaincomprising the same amino acid sequence as the heavy chain of theantibody produced by the hybridoma of ATCC® deposit number PTA-125005,e.g., in combination with a light chain.

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR and comprises a light chaincomprising the same amino acid sequence as the light chain of theantibody produced by the hybridoma of ATCC® deposit number PTA-125005,e.g., in combination with a heavy chain.

In certain aspects, an antibody or antigen-binding fragment thereofdescribed herein binds to human TTR and comprises a heavy chaincomprising the same amino acid sequence as the heavy chain of theantibody produced by the hybridoma of ATCC® deposit number PTA-125005,and a light chain comprising the same amino acid sequence as the lightchain of the antibody produced by the hybridoma of ATCC® deposit numberPTA-125005.

In specific aspects, provided herein are antibodies that comprise aheavy chain and alight chain.

In a specific aspect, the heavy chain of an antibody described herein isa gamma heavy chain (e.g., a human gamma heavy chain, e.g., humanIgG₁heavy chain). In a particular aspect, an antibody whichimmunospecifically binds to TTR (e.g., human TTR) provided hereincomprises a heavy chain wherein the amino acid sequence of the VH domaincomprises the VH sequence of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005, and wherein the constant region of theheavy chain comprises the amino acid sequence of a human gamma heavychain (e.g., human IgG₁heavy chain) constant region.

In a specific aspect, the light chain of an antibody described herein isa kappa light chain (e.g., a human kappa light chain). In a particularaspect, an antibody which immunospecifically binds to TTR (e.g., humanTTR) provided herein comprises a light chain wherein the amino acidsequence of the VL domain comprises the VL sequence of the antibodyproduced by the hybridoma of ATCC® deposit number PTA-125005, andwherein the constant region of the light chain comprises the amino acidsequence of a human kappa light chain constant region.

In a particular aspect, an antibody which immunospecifically binds toTTR (e.g., human TTR) provided herein comprises (i) a heavy chainwherein the amino acid sequence of the VH domain comprises the VHsequence of the antibody produced by the hybridoma of ATCC® depositnumber PTA-125005, and wherein the constant region of the heavy chaincomprises the amino acid sequence of a human gamma heavy chain (e.g.,human IgG₁heavy chain) constant region and (ii) comprises a light chainwherein the amino acid sequence of the VL domain comprises the VLsequence of the antibody produced by the hybridoma of ATCC® depositnumber PTA-125005, and wherein the constant region of the light chaincomprises the amino acid sequence of a human kappa light chain constantregion.

In another particular aspect, an antibody or antigen-binding fragmentthereof described herein, which immunospecifically binds to TTR (e.g.,human TTR), comprises a heavy chain and a light chain, wherein (i) theheavy chain comprises a VH domain comprising the CDR H1, CDR H2, and CDRH3 amino acid sequences of the CGX304 antibody listed in Table 1; (ii)the light chain comprises a VL domain comprising the CDR L1, CDR L2, andCDR L3 amino acid sequences of the CGX304 antibody listed in Table 2;(iii) the heavy chain further comprises a constant heavy chain domaincomprising the amino acid sequence of the constant domain of a humanIgG₁ heavy chain; and (iv) the light chain further comprises a constantlight chain domain comprising the amino acid sequence of the constantdomain of a human kappa light chain.

In another aspect, provided herein are antibody or antigen-bindingfragments thereof that specifically bind to TTR and decreases thetoxicity of TTR fibrils (e.g., on human cardiomyocytes). In anotheraspect, provided herein are antibodies or antigen-binding fragmentsthereof that specifically bind to TTR and inhibit accumulation of TTRaggregates in an organ. The organ can be, for example, a heart and/or akidney.

In another aspect, provided herein are antibodies or antigen-bindingfragments thereof that bind the same epitope of TTR (e.g., an epitope ofhuman TTR) as CGX304.

Competition binding assays can be used to determine whether twoantibodies bind to overlapping epitopes. Competitive binding can bedetermined in an assay in which the immunoglobulin under test inhibitsspecific binding of a reference antibody to a common antigen, such asTTR. Numerous types of competitive binding assays are known, forexample: solid phase direct or indirect radioimmunoassay (MA), solidphase direct or indirect enzyme immunoassay (EIA), sandwich competitionassay (see Stahli C et al., (1983) Methods Enzymol 9: 242-253); solidphase direct biotin-avidin EIA (see Kirkland T N et al., (1986) JImmunol 137: 3614-9); solid phase direct labeled assay, solid phasedirect labeled sandwich assay (see Harlow E & Lane D, (1988) Antibodies:A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labelRIA using I-125 label (see Morel G A et al., (1988) Mol Immunol 25(1):7-15); solid phase direct biotin-avidin EIA (Cheung R C et al., (1990)Virology 176: 546-52); and direct labeled MA. (Moldenhauer G et al.,(1990) Scand J Immunol 32: 77-82). Typically, such an assay involves theuse of purified antigen (e.g., TTR such as human TTR) bound to a solidsurface or cells bearing either of these, an unlabeled testimmunoglobulin and a labeled reference immunoglobulin. Competitiveinhibition can be measured by determining the amount of label bound tothe solid surface or cells in the presence of the test immunoglobulin.Usually the test immunoglobulin is present in excess. Usually, when acompeting antibody is present in excess, it will inhibit specificbinding of a reference antibody to a common antigen by at least 50-55%,55-60%, 60-65%, 65-70%, 70-75% or more. A competition binding assay canbe configured in a large number of different formats using eitherlabeled antigen or labeled antibody. In a common version of this assay,the antigen is immobilized on a 96-well plate. The ability of unlabeledantibodies to block the binding of labeled antibodies to the antigen isthen measured using radioactive or enzyme labels. For further detailssee, for example, Wagener C et al., (1983) J Immunol 130: 2308-2315;Wagener C et al., (1984) J Immunol Methods 68: 269-274; Kuroki M et al.,(1990) Cancer Res 50: 4872-4879; Kuroki M et al., (1992) Immunol Invest21: 523-538; Kuroki M et al., (1992) Hybridoma 11: 391-407 andAntibodies: A Laboratory Manual, Ed Harlow E & Lane D editors supra, pp.386-389.

In one aspect, a competition assay is performed using surface plasmonresonance (BIAcore®), e.g., by an ‘in tandem approach’ such as thatdescribed by Abdiche Y N et al., (2009) Analytical Biochem 386: 172-180,whereby TTR antigen is immobilized on the chip surface, for example, aCMS sensor chip and the anti-TTR antibodies are then run over the chip.To determine if an antibody or antigen-binding fragment thereof competeswith an anti-TTR antibody described herein, the anti-TTR antibody isfirst run over the chip surface to achieve saturation and then thepotential, competing antibody is added. Binding of the competingantibody or antigen-binding fragment thereof can then be determined andquantified relative to a non-competing control.

In one aspect, provided herein are antibodies that competitively inhibit(e.g., in a dose dependent manner) CGX304 from binding to TTR (e.g.,human TTR), as determined using assays known to one of skill in the artor described herein (e.g., ELISA competitive assays, or suspension arrayor surface plasmon resonance assay).

In specific aspects, provided herein is an antibody or antigen-bindingfragment which competitively inhibits (e.g., in a dose dependent manner)binding to TTR (e.g., human TTR), of an antibody comprising the VHsequence of the antibody produced by the hybridoma of ATCC® depositnumber PTA-125005, and the VL sequence of the antibody produced by thehybridoma of ATCC® deposit number PTA-125005.

In a specific aspect, an antigen-binding fragment as described herein,which immunospecifically binds to TTR (e.g., human TTR), is selectedfrom the group consisting of a Fab, Fab′, F(ab′)₂, and scFv, wherein theFab, Fab′, F(ab′)₂, or scFv comprises a heavy chain variable regionsequence and a light chain variable region sequence of an anti-TTRantibody or antigen-binding fragment thereof as described herein. A Fab,Fab′, F(ab′)2, or scFv can be produced by any technique known to thoseof skill in the art. In certain aspects, the Fab, Fab′, F(ab′)₂, or scFvfurther comprises a moiety that extends the half-life of the antibody invivo. The moiety is also termed a “half-life extending moiety.” Anymoiety known to those of skill in the art for extending the half-life ofa Fab, Fab′, F(ab′)₂, or scFv in vivo can be used. For example, thehalf-life extending moiety can include a Fc region, a polymer, analbumin, or an albumin binding protein or compound. The polymer caninclude a natural or synthetic, optionally substituted straight orbranched chain polyalkylene, polyalkenylene, polyoxylalkylene,polysaccharide, polyethylene glycol, polypropylene glycol, polyvinylalcohol, methoxypolyethylene glycol, lactose, amylose, dextran,glycogen, or derivative thereof. Substituents can include one or morehydroxy, methyl, or methoxy groups. In certain aspects, the Fab, Fab′,F(ab′)₂, or scFv can be modified by the addition of one or moreC-terminal amino acids for attachment of the half-life extending moiety.In certain aspects the half-life extending moiety is polyethylene glycolor human serum albumin. In certain aspects, the Fab, Fab′, F(ab′)₂, orscFv is fused to an Fc region.

An anti-TTR antibody or antigen-binding fragment thereof can be fused orconjugated (e.g., covalently or noncovalently linked) to a detectablelabel or substance. Examples of detectable labels or substances includeenzyme labels, such as, glucose oxidase; radioisotopes, such as iodine(¹²⁵I, ¹²¹I) carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (¹²¹In),and technetium (⁹⁹Tc); luminescent labels, such as luminol; andfluorescent labels, such as fluorescein and rhodamine, and biotin. Suchlabeled antibodies or antigen-binding fragments thereof can be used todetect TTR (e.g., human TTR) protein.

III. Antibody Production

Antibodies and antigen-binding fragments thereof that immunospecificallybind to TTR (e.g., human TTR) can be produced by any method known in theart for the synthesis of antibodies and antigen-binding fragmentsthereof, for example, by chemical synthesis or by recombinant expressiontechniques. The methods described herein employ, unless otherwiseindicated, conventional techniques in molecular biology, microbiology,genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR,oligonucleotide synthesis and modification, nucleic acid hybridization,and related fields within the skill of the art. These techniques aredescribed, for example, in the references cited herein and are fullyexplained in the literature. See, e.g., Sambrook J et al., (2001)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y.; Ausubel F M et al., Current Protocolsin Molecular Biology, John Wiley & Sons (1987 and annual updates);Current Protocols in Immunology, John Wiley & Sons (1987 and annualupdates) Gait (ed.) (1984) Oligonucleotide Synthesis: A PracticalApproach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides andAnalogues: A Practical Approach, IRL Press; Birren B et al., (eds.)(1999) Genome Analysis: A Laboratory Manual, Cold Spring HarborLaboratory Press.

In a certain aspect, provided herein is a method of making an antibodyor antigen-binding fragment which immunospecifically binds to TTR (e.g.,human TTR) comprising culturing a cell or host cell described herein. Ina certain aspect, provided herein is a method of making an antibody orantigen-binding fragment thereof which immunospecifically binds to TTR(e.g., human TTR) comprising expressing (e.g., recombinantly expressing)the antibody or antigen-binding fragment thereof using a cell or hostcell described herein (e.g., a cell or a host cell comprisingpolynucleotides encoding an antibody or antigen-binding fragment thereofdescribed herein). In a particular aspect, the cell is an isolated cell.In a particular aspect, the exogenous polynucleotides have beenintroduced into the cell. In a particular aspect, the method furthercomprises the step of purifying the antibody or antigen-binding fragmentobtained from the cell or host cell.

Methods for producing polyclonal antibodies are known in the art (see,for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002)5th Ed., Ausubel F M et al., eds., John Wiley and Sons, New York).

Monoclonal antibodies or antigen-binding fragments thereof can beprepared using a wide variety of techniques known in the art includingthe use of hybridoma, recombinant, and phage display technologies,yeast-based presentation technologies, or a combination thereof. Forexample, monoclonal antibodies or antigen-binding fragments thereof canbe produced using hybridoma techniques including those known in the artand taught, for example, in Harlow E & Lane D, Antibodies: A LaboratoryManual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); HammerlingG J et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681(Elsevier, N.Y., 1981), or as described in Kohler G & Milstein C (1975)Nature 256: 495. Examples of yeast-based presentation methods that canbe employed to select and generate the antibodies described hereininclude those disclosed in, for example, WO2009/036379A2; WO2010/105256;and WO2012/009568, each of which is herein incorporated by reference inits entirety.

In specific aspects, a monoclonal antibody or antigen-binding fragmentthereof may be produced using the hybridoma method first described byKohler et al., Nature, 256:495 (1975), as mentioned above. In thehybridoma method, a mouse or another appropriate host animal isimmunized as above described to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theprotein used for immunization, for example, variant mixtures of TTR.Lymphocytes then are fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell (Goding,Monoclonal Antibodies: Principles and Practice, pp. 59-103 (AcademicPress, 1986).

In specific aspects, a monoclonal antibody or antigen-binding fragmentthereof is an antibody or antigen-binding fragment produced by a clonalcell (e.g., hybridoma or host cell producing a recombinant antibody orantigen-binding fragment), wherein the antibody or antigen-bindingfragment immunospecifically binds to TTR (e.g., human TTR) asdetermined, e.g., by ELISA or other antigen-binding assays known in theart or in the Examples provided herein. In particular aspects, amonoclonal antibody or antigen-binding fragment thereof can be a rodentor murine antibody or antigen-binding fragment thereof. In particularaspects, a monoclonal antibody or antigen-binding fragment thereof canbe a chimeric or a humanized antibody or antigen-binding fragmentthereof. In certain aspects, a monoclonal antibody or antigen-bindingfragment thereof can be a Fab fragment or an F(ab′)₂ fragment.Monoclonal antibodies or antigen-binding fragments thereof describedherein can, for example, be made by the hybridoma method as described inKohler G & Milstein C (1975) Nature 256: 495 or can, e.g., be isolatedfrom phage libraries using the techniques as described herein, forexample. Other methods for the preparation of clonal cell lines and ofmonoclonal antibodies and antigen-binding fragments thereof expressedthereby are well known in the art (see, for example, Chapter 11 in:Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel F M etal., supra).

Antigen-binding fragments of antibodies described herein can begenerated by any technique known to those of skill in the art. Forexample, Fab and F(ab′)₂ fragments described herein can be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). A Fab fragment corresponds to one of the two identical armsof a tetrameric antibody molecule and contains the complete light chainpaired with the VH and CH1 domains of the heavy chain. An F(ab′)₂fragment contains the two antigen-binding arms of a tetrameric antibodymolecule linked by disulfide bonds in the hinge region.

Further, the antibodies or antigen-binding fragments thereof describedherein can also be generated using various phage display and/oryeast-based presentation methods known in the art. In phage displaymethods, proteins are displayed on the surface of phage particles whichcarry the polynucleotide sequences encoding them. In particular, DNAsequences encoding VH and VL domains are amplified from animal cDNAlibraries (e.g., human or murine cDNA libraries of affected tissues).The DNA encoding the VH and VL domains are recombined together with ascFv linker by PCR and cloned into a phagemid vector. The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13, and the VH and VL domains are usually recombinantly fused toeither the phage gene III or gene VIII. Phage expressing an antibody orantigen-binding fragment thereof that binds to a particular antigen canbe selected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Examples of phagedisplay methods that can be used to make the antibodies or fragmentsdescribed herein include those disclosed in Brinkman U et al., (1995) JImmunol Methods 182: 41-50; Ames R S et al., (1995) J Immunol Methods184: 177-186; Kettleborough C A et al., (1994) Eur J Immunol 24:952-958; Persic L et al., (1997) Gene 187: 9-18; Burton D R & Barbas C F(1994) Advan Immunol 57: 191-280; PCT Application No. PCT/GB91/001134;International Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047, WO92/18619, WO 93/1 1236, WO 95/15982, WO 95/20401, and WO 97/13844; andU.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,5,658,727, 5,733,743, and 5,969,108.

An antibody or antigen-binding fragment thereof can be selected from anyclass of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and anyisotype, including IgG₁, IgG₂, IgG₃ and IgG₄.

IIIa. Polynucleotides

In certain aspects, provided herein are polynucleotides comprising anucleotide sequence encoding an antibody or antigen-binding fragmentthereof described herein or a domain thereof (e.g., a variable lightchain region and/or variable heavy chain region) that immunospecificallybinds to a TTR (e.g., human TTR) antigen, and vectors, e.g., vectorscomprising such polynucleotides for recombinant expression in host cells(e.g., E. coli and mammalian cells).

In particular aspects, provided herein are polynucleotides comprisingnucleotide sequences encoding antibodies or antigen-binding fragmentsthereof, which immunospecifically bind to a TTR polypeptide (e.g., humanTTR) and comprise an amino acid sequence as described herein, as well asantibodies or antigen-binding fragments that compete with suchantibodies or antigen-binding fragments for binding to a TTR polypeptide(e.g., in a dose-dependent manner), or which bind to the same epitope asthat of such antibodies or antigen-binding fragments.

Also provided herein is a polynucleotide comprising the same amino acidsequence as the VH of the antibody produced by the hybridoma of ATCC®deposit number PTA-125005. In some aspects, an antibody orantigen-binding fragment thereof comprising the polypeptideimmunospecifically binds to TTR.

Also provided herein is a polynucleotide comprising the same amino acidsequence as the VL of the antibody produced by the hybridoma of ATCC®deposit number PTA-125005. In some aspects, an antibody orantigen-binding fragment thereof comprising the polypeptideimmunospecifically binds to TTR.

Also provided herein is a polynucleotide comprising a nucleotidesequence encoding a polypeptide comprising a sequence selected from thegroup consisting of SEQ ID NOs:1-3 or comprising the amino acids of allof SEQ ID NOs:1-3. In some aspects, an antibody or antigen-bindingfragment thereof comprising the polypeptide immunospecifically binds toTTR. Also provided herein is a polynucleotide comprising a nucleotidesequence encoding a polypeptide comprising a sequence selected from thegroup consisting of SEQ ID NOs:4-6 or comprising all of SEQ ID NOs:4-6.In some aspects, an antibody or antigen-binding fragment thereofcomprising the polypeptide immunospecifically binds to TTR.

Also provided herein is a polynucleotide comprising a nucleotidesequence encoding a polypeptide comprising a sequence selected from thegroup consisting of SEQ ID NOs:8-10 or comprising the amino acids of allof SEQ ID NOs:8-10. In some aspects, an antibody or antigen-bindingfragment thereof comprising the polypeptide immunospecifically binds toTTR. Also provided herein is a polynucleotide comprising a nucleotidesequence encoding a polypeptide comprising a sequence selected from thegroup consisting of SEQ ID NOs:11, 12, and 6 or comprising all of SEQ IDNOs:11, 12, and 6. In some aspects, an antibody or antigen-bindingfragment thereof comprising the polypeptide immunospecifically binds toTTR.

Also provided herein is a polynucleotide comprising a nucleotidesequence encoding a heavy chain polypeptide comprising the same aminoacid sequence as the heavy chain of the antibody produced by thehybridoma of ATCC® deposit number PTA-125005. In some aspects, anantibody or antigen-binding fragment thereof comprising the polypeptideimmunospecifically binds to TTR.

Also provided herein is a polynucleotide comprising the same amino acidsequence as the light chain of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005. In some aspects, an antibody orantigen-binding fragment thereof comprising the polypeptideimmunospecifically binds to TTR.

A nucleic acid encoding a heavy chain variable domain or heavy chain anda nucleic acid encoding a light chain variable domain or light chain maybe in the same polynucleotide or in different polynucleotides. A nucleicacid encoding a heavy chain variable domain or heavy chain and a nucleicacid encoding a light chain variable domain or light chain may be in thesame vector or in different vectors.

Also provided herein is a composition comprising (i) a polynucleotidecomprising a nucleic acid encoding a heavy chain variable domain,wherein the nucleic acid encoding the heavy chain variable domaincomprises the heavy chain variable domain-encoding sequence in thehybridoma of ATCC® deposit number PTA-125005, and (ii) a polynucleotidecomprising a nucleic acid encoding a light chain variable domain,wherein the nucleic acid encoding the light chain variable domaincomprises the light chain variable domain-encoding sequence in thehybridoma of ATCC® deposit number PTA-125005. The nucleic acid encodingthe heavy chain variable domain and the nucleic acid encoding the lightchain variable domain may be in the same polynucleotide or in differentpolynucleotides. The nucleic acid encoding the heavy chain variabledomain and the nucleic acid encoding the light chain variable domain maybe in the same vector or in different vectors.

Also provided herein are polynucleotides comprising a nucleotidesequence that encodes SEQ ID NOs:1-3 and/or 4-6. Also provided hereinare polynucleotides comprising a nucleotide sequence that encodes SEQ IDNOs:8-10 and/or 11, 12, and 6.

Also provided herein are polynucleotides that are at least about 80%,85%, or 90% identical to a nucleotide sequence that encodes SEQ IDNOs:1-3 and/or 4-6. Also provided herein are polynucleotides that are atleast about 80%, 85%, or 90% identical to a nucleotide sequence thatencodes SEQ ID NOs:8-10 and/or 11, 12, and 6.

Also provided herein are polynucleotides comprising a nucleotidesequence that is at least about 95% identical to a nucleotide sequencethat encodes SEQ ID NOs:1-3 and/or 4-6. Also provided herein arepolynucleotides comprising a nucleotide sequence that is at least about96% identical to a nucleotide sequence that encodes SEQ ID NOs:1-3and/or 4-6. Also provided herein are polynucleotides comprising anucleotide sequence that is at least about 97% identical to a nucleotidesequence that encodes SEQ ID NOs:1-3 and/or 4-6. Also provided hereinare polynucleotides comprising a nucleotide sequence that is at leastabout 98% identical to a nucleotide sequence that encodes SEQ ID NOs:1-3and/or 4-6. Also provided herein are polynucleotides comprising anucleotide sequence that is at least about 99% identical to a nucleotidesequence that encodes SEQ ID NOs:1-3 and/or 4-6.

Also provided herein are polynucleotides comprising a nucleotidesequence that is at least about 95% identical to a nucleotide sequencethat encodes SEQ ID NOs:8-10 and/or 11, 12, and 6. Also provided hereinare polynucleotides comprising a nucleotide sequence that is at leastabout 96% identical to a nucleotide sequence that encodes SEQ ID NOs:8-10 and/or 11, 12, and 6. Also provided herein are polynucleotidescomprising a nucleotide sequence that is at least about 97% identical toa nucleotide sequence that encodes SEQ ID NOs: 8-10 and/or 11, 12, and6. Also provided herein are polynucleotides comprising a nucleotidesequence that is at least about 98% identical to a nucleotide sequencethat encodes SEQ ID NOs: 8-10 and/or 11, 12, and 6. Also provided hereinare polynucleotides comprising a nucleotide sequence that is at leastabout 99% identical to a nucleotide sequence that encodes SEQ ID NOs:8-10 and/or 11, 12, and 6.

In a particular aspect, a polynucleotide or combination ofpolynucleotides provided herein comprises a nucleotide sequence orcombination of nucleotide sequences encoding an antibody orantigen-binding fragment thereof that immunospecifically binds to TTR(e.g., human TTR), wherein the antibody or antigen-binding fragmentthereof comprises a heavy chain, wherein the heavy chain comprises thesame amino acid sequence as the VH of the antibody produced by thehybridoma of ATCC® deposit number PTA-125005, and a constant regioncomprising the amino acid sequence of a human gamma (γ) heavy chainconstant region (e.g., IgG1).

In a particular aspect, a polynucleotide or combination ofpolynucleotides provided herein comprises a nucleotide sequence orcombination of nucleotide sequences encoding an antibody orantigen-binding fragment thereof that immunospecifically binds to TTR(e.g., human TTR), wherein the antibody or antigen-binding fragmentthereof comprises a light chain, wherein the light chain comprises thesame amino acid sequence as the VL of the antibody produced by thehybridoma of ATCC® deposit number PTA-125005, and a constant regioncomprising the amino acid sequence of a human kappa light chain constantregion.

Also provided herein are polynucleotides encoding an anti-TTR antibodyor antigen-binding fragment thereof described herein or a domain thereofthat are optimized, e.g., by codon/RNA optimization, replacement withheterologous signal sequences, and elimination of mRNA instabilityelements. Methods to generate optimized nucleic acids encoding ananti-TTR antibody or antigen-binding fragment thereof or a domainthereof (e.g., heavy chain, light chain, VH domain, or VL domain) forrecombinant expression by introducing codon changes (e.g., a codonchange that encodes the same amino acid due to the degeneracy of thegenetic code) and/or eliminating inhibitory regions in the mRNA can becarried out by adapting the optimization methods described in, e.g.,U.S. Pat. Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and6,794,498, accordingly.

A polynucleotide encoding an antibody or antigen-binding fragmentthereof described herein or a domain thereof can be generated fromnucleic acid from a suitable source (e.g., a hybridoma) using methodswell known in the art (e.g., PCR and other molecular cloning methods).For example, PCR amplification using synthetic primers hybridizable tothe 3′ and 5′ ends of a known sequence can be performed using genomicDNA obtained from hybridoma cells producing the antibody of interest.Such PCR amplification methods can be used to obtain nucleic acidscomprising the sequence encoding the light chain and/or heavy chain ofan antibody or antigen-binding fragment thereof. Such PCR amplificationmethods can be used to obtain nucleic acids comprising the sequenceencoding the variable light chain region and/or the variable heavy chainregion of an antibody or antigen-binding fragment thereof. The amplifiednucleic acids can be cloned into vectors for expression in host cellsand for further cloning, for example, to generate chimeric and humanizedantibodies or antigen-binding fragments thereof.

Polynucleotides provided herein can be, e.g., in the form of RNA or inthe form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, andDNA can be double-stranded or single-stranded. If single stranded, DNAcan be the coding strand or non-coding (anti-sense) strand. In certainaspects, the polynucleotide is a cDNA or a DNA lacking one moreendogenous introns. In certain aspects, a polynucleotide is anon-naturally occurring polynucleotide. In certain aspects, apolynucleotide is recombinantly produced. In certain aspects, thepolynucleotides are isolated. In certain aspects, the polynucleotidesare substantially pure. In certain aspects, a polynucleotide is purifiedfrom natural components.

IIIb. Cells and Vectors

In certain aspects, provided herein are vectors (e.g., expressionvectors) comprising polynucleotides comprising nucleotide sequencesencoding anti-TTR antibodies and antigen-binding fragments thereof or adomain thereof for recombinant expression in host cells, preferably inmammalian cells. Also provided herein are cells, e.g., host cells,comprising such vectors for recombinantly expressing anti-TTR antibodiesor antigen-binding fragments thereof described herein. In a particularaspect, provided herein are methods for producing an antibody orantigen-binding fragments thereof described herein, comprisingexpressing such antibody or antigen-binding fragment thereof in a hostcell.

In certain aspects, recombinant expression of an antibody orantigen-binding fragment thereof or domain thereof described herein(e.g., a heavy or light chain described herein) that specifically bindsto TTR (e.g., human TTR) involves construction of an expression vectorcontaining a polynucleotide that encodes the antibody or antigen-bindingfragment thereof or domain thereof. Once a polynucleotide encoding anantibody or antigen-binding fragment thereof or domain thereof (e.g.,heavy or light chain variable domain) described herein has beenobtained, the vector for the production of the antibody orantigen-binding fragment thereof can be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody or antigen-binding fragment thereof or domain thereof (e.g.,light chain or heavy chain) encoding nucleotide sequence are describedherein. Methods which are well known to those skilled in the art can beused to construct expression vectors containing antibody orantigen-binding fragment thereof or domain thereof (e.g., light chain orheavy chain) coding sequences and appropriate transcriptional andtranslational control signals. These methods include, for example, invitro recombinant DNA techniques, synthetic techniques, and in vivogenetic recombination. Also provided are replicable vectors comprising anucleotide sequence encoding an antibody or antigen-binding fragmentthereof described herein, a heavy or light chain, a heavy or light chainvariable domain, or a heavy or light chain CDR, operably linked to apromoter. Such vectors can, for example, include the nucleotide sequenceencoding the constant region of the antibody or antigen-binding fragmentthereof (see, e.g., International Publication Nos. WO 86/05807 and WO89/01036; and U.S. Pat. No. 5,122,464), and variable domains of theantibody or antigen-binding fragment thereof can be cloned into such avector for expression of the entire heavy, the entire light chain, orboth the entire heavy and light chains.

An expression vector can be transferred to a cell (e.g., host cell) byconventional techniques, and the resulting cells can then be cultured byconventional techniques to produce an antibody or antigen-bindingfragment thereof described herein, e.g., an antibody or antigen-bindingfragment thereof comprising the six CDRs of SEQ ID NOs:1-6, the six CDRSof SEQ ID NOs:8-12 and 6, the same amino acid sequence as the VH of theantibody produced by the hybridoma of ATCC® deposit number PTA-125005,the same amino acid sequence as the VL of the antibody produced by thehybridoma of ATCC® deposit number PTA-125005, the same amino acidsequence as the VH of the antibody produced by the hybridoma of ATCC®deposit number PTA-125005, and the same amino acid sequence as the VL ofthe antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, the same amino acid sequence as the heavy chain of theantibody produced by the hybridoma of ATCC® deposit number PTA-125005,the same amino acid sequence as the light chain of the antibody producedby the hybridoma of ATCC® deposit number PTA-125005, or the same aminoacid sequence as the heavy chain of the antibody produced by thehybridoma of ATCC® deposit number PTA-125005, and the same amino acidsequence as the light chain of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005. Thus, provided herein are host cellscontaining a polynucleotide encoding an antibody or antigen-bindingfragment thereof described herein, e.g., an antibody or antigen-bindingfragment thereof comprising the six CDRs of SEQ ID NOs:1-6, the six CDRsof SEQ ID NOs:8-12 and 6, the same amino acid sequence as the VH of theantibody produced by the hybridoma of ATCC® deposit number PTA-125005,the same amino acid sequence as the VL of the antibody produced by thehybridoma of ATCC® deposit number PTA-125005, the same amino acidsequence as the VH of the antibody produced by the hybridoma of ATCC®deposit number PTA-125005, and the same amino acid sequence as the VL ofthe antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, the same amino acid sequence as the heavy chain of theantibody produced by the hybridoma of ATCC® deposit number PTA-125005,the same amino acid sequence as the light chain of the antibody producedby the hybridoma of ATCC® deposit number PTA-125005, or the same aminoacid sequence as the heavy chain of the antibody produced by thehybridoma of ATCC® deposit number PTA-125005 and the same amino acidsequence as the light chain of the antibody produced by the hybridoma ofATCC® deposit number PTA-125005, operably linked to a promoter forexpression of such sequences in the host cell. In certain aspects, forthe expression of double-chained antibodies or antigen-binding fragmentsthereof, vectors encoding both the heavy and light chains, individually,can be co-expressed in the host cell for expression of the entireimmunoglobulin, as detailed below. In certain aspects, a host cellcontains a vector comprising a polynucleotide encoding both the heavychain and light chain of an antibody described herein or a domainthereof. In specific aspects, a host cell contains two differentvectors, a first vector comprising a polynucleotide encoding a heavychain or a heavy chain variable region of an antibody or antigen-bindingfragment thereof described herein, and a second vector comprising apolynucleotide encoding a light chain or a light chain variable regionof an antibody described herein (e.g., an antibody comprising the sixCDRs of SEQ ID NOs:1-6 or the six CDRs of SEQ ID NOs:8-12 and 6), or adomain thereof. In other aspects, a first host cell comprises a firstvector comprising a polynucleotide encoding a heavy chain or a heavychain variable region of an antibody or antigen-binding fragment thereofdescribed herein, and a second host cell comprises a second vectorcomprising a polynucleotide encoding a light chain or a light chainvariable region of an antibody or antigen-binding fragment thereofdescribed herein (e.g., an antibody or antigen-binding fragment thereofcomprising the six CDRs of SEQ ID NOs:1-6 or the six CDRS of SEQ IDNOs:8-12 and 6). In specific aspects, a heavy chain/heavy chain variableregion expressed by a first cell associated with a light chain/lightchain variable region of a second cell to form an anti-TTR antibody orantigen-binding fragment thereof described herein (e.g., antibody orantigen-binding fragment thereof comprising the six CDRs of SEQ IDNOs:1-6 or the six CDRS of SEQ ID NOs:8-12 and 6). In certain aspects,provided herein is a population of host cells comprising such first hostcell and such second host cell.

In a particular aspect, provided herein is a population of vectorscomprising a first vector comprising a polynucleotide encoding a lightchain/light chain variable region of an anti-TTR antibody orantigen-binding fragment thereof described herein, and a second vectorcomprising a polynucleotide encoding a heavy chain/heavy chain variableregion of an anti-TTR antibody or antigen-binding fragment thereofdescribed herein (e.g., antibody or antigen-binding fragment thereofcomprising the CDRs of SEQ ID NOs:1-6 or the CDRS of SEQ ID NOs:8-12 and6). Alternatively, a single vector can be used which encodes, and iscapable of expressing, both heavy and light chain polypeptides.

A variety of host-expression vector systems can be utilized to expressantibodies and antigen-binding fragments thereof described herein (e.g.,an antibody or antigen-binding fragment thereof comprising the CDRs ofSEQ ID NOs:1-6 or the CDRS of SEQ ID NOs:8-12 and 6) (see, e.g., U.S.Pat. No. 5,807,715). Such host-expression systems represent vehicles bywhich the coding sequences of interest can be produced and subsequentlypurified, but also represent cells which can, when transformed ortransfected with the appropriate nucleotide coding sequences, express anantibody or antigen-binding fragment thereof described herein in situ.These include but are not limited to microorganisms such as bacteria(e.g., E. coli and B. subtilis) transformed with recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorscontaining antibody coding sequences; yeast (e.g., Saccharomyces Pichia)transformed with recombinant yeast expression vectors containingantibody coding sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems (e.g., green algae such as Chlamydomonasreinhardtii) infected with recombinant virus expression vectors (e.g.,cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing antibody coding sequences; or mammalian cell systems(e.g., COS (e.g., COS1 or COS), CHO (e.g., CHO-K1 SP), BHK, MDCK, HEK293, NSO, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T,HepG2, SP210, R1.1, B-W, L-M, BSC1, BSC40, YB/20 and BMT10 cells)harboring recombinant expression constructs containing promoters derivedfrom the genome of mammalian cells (e.g., metallothionein promoter) orfrom mammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). In a specific aspect, cells for expressingantibodies and antigen-binding fragments thereof described herein (e.g.,an antibody or antigen-binding fragment thereof comprising the CDRs ofSEQ ID NOs:1-6 or the CDRS of SEQ ID NOs:8-12 and 6) are CHO cells, forexample CHO cells from the CHO GS System™ (Lonza) or CHO-K1 SP cells. Ina particular aspect, cells for expressing antibodies described hereinare human cells, e.g., human cell lines. In a specific aspect, amammalian expression vector is pOptiVEC™ or pcDNA3.3. In a particularaspect, bacterial cells such as Escherichia coli, or eukaryotic cells(e.g., mammalian cells), especially for the expression of wholerecombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary (CHO) cells in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking M K & Hofstetter H (1986) Gene 45: 101-105; and Cockett M I etal., (1990) Biotechnology 8: 662-667). In certain aspects, antibodies orantigen-binding fragments thereof described herein are produced by CHOcells or NSO cells.

In addition, a host cell strain can be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products cancontribute to the function of the protein. To this end, eukaryotic hostcells that possess the cellular machinery for proper processing of theprimary transcript, glycosylation, and phosphorylation of the geneproduct can be used. Such mammalian host cells include but are notlimited to CHO, VERO, BHK, HeLa, MDCK, HEK 293, NIH 3T3, W138, BT483,Hs578T, HTB2, BT2O and T47D, NSO (a murine myeloma cell line that doesnot endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g.,COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, B-W,L-M, BSC1, BSC40, YB/20, BMT10 and HsS78Bst cells. In certain aspects,anti-TTR antibodies described herein (e.g., an antibody orantigen-binding fragment thereof comprising the CDRs of SEQ ID NOs:1-6or the CDRS of SEQ ID NOs:8-12 and 6) are produced in mammalian cells,such as CHO cells, e.g., CHO-K1 or CHO-K1 SP cells. In certain aspects,anti-TTR antibodies described herein (e.g., an antibody orantigen-binding fragment thereof comprising the CDRs of SEQ ID NOs:1-6or the CDRS of SEQ ID NOs:8-12 and 6) are produced in mammalian cells,such as HEK-293 cells, e.g., 293F cells.

In some aspects, a signal peptide is used in constructing a vectorcontaining the VH and/or VL or the heavy and/or light chain of anantibody or antigen-binding fragment thereof provided herein.

Once an antibody or antigen-binding fragment thereof described hereinhas been produced by recombinant expression, it can be purified by anymethod known in the art for purification of an immunoglobulin molecule,for example, by chromatography (e.g., ion exchange, affinity,particularly by affinity for the specific antigen after Protein A, andsizing column chromatography), centrifugation, differential solubility,or by any other standard technique for the purification of proteins.Further, the antibodies or antigen-binding fragments thereof describedherein can be fused to heterologous polypeptide sequences describedherein or otherwise known in the art to facilitate purification.

In specific aspects, an antibody or antigen-binding fragment thereofdescribed herein is isolated or purified. Generally, an isolatedantibody or antigen-binding fragment thereof is one that issubstantially free of other antibodies or antigen-binding fragmentsthereof with different antigenic specificities than the isolatedantibody or antigen-binding fragment thereof. For example, in aparticular aspect, a preparation of an antibody or antigen-bindingfragment thereof described herein is substantially free of cellularmaterial and/or chemical precursors.

IV. Pharmaceutical Compositions Comprising Anti-TTR Antibodies andAntigen-Binding Fragments Thereof

Provided herein are compositions comprising an antibody orantigen-binding fragment thereof described herein having the desireddegree of purity in a physiologically acceptable carrier, excipient orstabilizer (Remington's Pharmaceutical Sciences (1990) Mack PublishingCo., Easton, Pa.). Acceptable carriers, excipients, or stabilizers arenontoxic to recipients at the dosages and concentrations employed.

In various aspects, compositions comprising an anti-TTR antibody orantigen-binding fragment thereof are provided in formulations with apharmaceutically acceptable carrier (see, e.g., Gennaro, Remington: TheScience and Practice of Pharmacy with Facts and Comparisons: DrugfactsPlus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms andDrug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004);Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed.,Pharmaceutical Press (2000)).

Pharmaceutical compositions described herein can be useful in decreasingthe toxicity of TTR fibrils (e.g., on human cardiomyocytes) and/orinhibiting accumulation of TTR aggregates (e.g., in an organ such as aheart and/or kidney). Pharmaceutical compositions described herein canbe useful in treating a condition such as a TTR amyloidosis.

The pharmaceutical compositions described herein are in one aspect foruse as a medicament. The pharmaceutical compositions described hereinare in one aspect for use as a diagnostic, e.g., to detect the presenceof TTR in a sample obtained from a patient (e.g., a human patient).

The compositions to be used for in vivo administration can be sterile.This is readily accomplished by filtration through, e.g., sterilefiltration membranes.

In some aspects, pharmaceutical compositions are provided, wherein thepharmaceutical composition comprises anti-TTR antibodies orantigen-binding fragments thereof described herein and apharmaceutically acceptable carrier.

In some aspects, a pharmaceutical composition comprises (i) an isolatedantibody or antigen-binding fragment thereof that specifically binds tohuman TTR, comprising (a) the complementarity determining region (CDR)H1, CDR H2, CDR H3 and CDR L1, CDR L2, and CDR L3 sequences of SEQ IDNOs:1-6, respectively, (b) the CDR H1, CDR H2, CDR H3 and CDR L1, CDRL2, and CDR L3 sequences of SEQ ID NOs:8-12 and 6, respectively, (c) thesame amino acid sequence as the VH of the antibody produced by thehybridoma of ATCC® deposit number PTA-125005, and the same amino acidsequence as the VL of the antibody produced by the hybridoma of ATCC®deposit number PTA-125005, or (d) the same amino acid sequence as theheavy chain of the antibody produced by the hybridoma of ATCC® depositnumber PTA-125005, and the same amino acid sequence as the light chainof the antibody produced by the hybridoma of ATCC® deposit numberPTA-125005, and (ii) a pharmaceutically acceptable excipient.

V. Methods of Using Anti-TTR Antibodies and Antigen-Binding FragmentsThereof

Va. Therapeutic and Prophylactic Uses and Methods

In one aspect, presented herein are methods for treating or preventing aTTR amyloidosis in a subject, comprising administering to a subject inneed thereof an anti-TTR antibody or antigen-binding fragment thereofdescribed herein, or a pharmaceutical composition thereof as describedabove and herein.

In another aspect, an anti-TTR antibody or antigen-binding fragmentthereof, or pharmaceutical composition, is administered to a patient(e.g., a human patient) to decrease the toxicity of TTR fibrils (e.g.,on human cardiomyocytes). In another aspect, an anti-TTR antibody orantigen-binding fragment thereof, or pharmaceutical composition, isadministered to a patient (e.g., a human patient) to inhibitaccumulation of TTR aggregates (e.g., in an organ such as a heart and/orkidney).

Usually, the patient is a human, but non-human mammals includingtransgenic mammals can also be treated.

In some aspects, the present invention relates to an antibody orantigen-binding fragment thereof or pharmaceutical composition providedherein for use as a medicament. In some aspects, the present inventionrelates to an antibody or antigen-binding fragment thereof orpharmaceutical composition provided herein, for use in a method for thetreatment or prevention of a TTR amyloidosis. In some aspects, thepresent invention relates to an antibody or antigen-binding fragmentthereof or pharmaceutical composition provided herein, for use in amethod for the treatment or prevention of a TTR amyloidosis in asubject, comprising administering to the subject an effective amount ofan antibody or antigen-binding fragment thereof or pharmaceuticalcomposition provided herein.

An antibody or antigen-binding fragment thereof or composition describedherein can be delivered to a subject by a variety of routes, such asparenteral, subcutaneous, intravenous, intradermal, transdermal, andintranasal. In one aspect, the antibody or antigen-binding fragmentthereof or composition is administered by an intravenous route.

The amount of an antibody or antigen-binding fragment thereof orcomposition which will be effective in the treatment or prevention of acondition will depend on the nature of the disease. The precise dose tobe employed in a composition will also depend on the route ofadministration, and the seriousness of the disease.

Vb. Detection and Diagnostic Uses

An anti-TTR antibody or antigen-binding fragment thereof describedherein can be used to assay TTR protein levels in a biological sampleusing classical methods known to those of skill in the art, includingimmunoassays, such as the enzyme linked immunosorbent assay (ELISA),fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC),immunoprecipitation, and Western blotting. Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹²⁵I, ¹²¹J) carbon (¹⁴C), sulfur(³⁵S), tritium (³H), indium (¹²¹In), and technetium (⁹⁹Tc); luminescentlabels, such as luminol; and fluorescent labels, such as fluorescein andrhodamine, and biotin. Such labels can be used to label an antibody orantigen-binding fragment thereof described herein. Alternatively, asecond antibody or antigen-binding fragment thereof that recognizes ananti-TTR antibody or antigen-binding fragment thereof described hereincan be labeled and used in combination with an anti-TTR antibody orantigen-binding fragment thereof to detect TTR protein levels.

Assaying for the expression level of TTR protein is intended to includequalitatively or quantitatively measuring or estimating the level of aTTR protein in a first biological sample either directly (e.g., bydetermining or estimating absolute protein level) or relatively (e.g.,by comparing to the disease associated protein level in a secondbiological sample). TTR polypeptide expression level in the firstbiological sample can be measured or estimated and compared to astandard TTR protein level, the standard being taken from a secondbiological sample obtained from an individual not having thedisorderorbeingdeterminedbyaveraginglevelsfromapopulationofindividualsnothavingthe disorder. As will be appreciated in the art, once the “standard” TTRpolypeptide level is known, it can be used repeatedly as a standard forcomparison.

As used herein, the term “biological sample” refers to any biologicalsample obtained from a subject, cell line, tissue, or other source ofcells potentially expressing TTR. Methods for obtaining tissue biopsiesand body fluids from animals (e.g., humans) are well known in the art. Abiological sample may also be a blood sample.

An anti-TTR antibody or antigen-binding fragment thereof describedherein can be used for prognostic, diagnostic, monitoring and screeningapplications, including in vitro and in vivo applications well known andstandard to the skilled artisan and based on the present description.Prognostic, diagnostic, monitoring and screening assays and kits for invitro assessment and evaluation of TTR presence may be utilized topredict, diagnose and monitor to evaluate patient samples includingthose known to have or suspected of having a TTR amyloidosis. This typeof prognostic and diagnostic monitoring and assessment is already inpractice utilizing antibodies against the HER2 protein in breast cancer(HercepTest™, Dako) where the assay is also used to evaluate patientsfor antibody therapy using Herceptin®. In vivo applications includedirected cell therapy and immune system modulation and radio imaging ofimmune responses.

Anti-TTR antibodies and antigen-binding fragments thereof describedherein can carry a detectable or functional label.

Examples of detectable moieties that can be used herein include but arenot limited to radioactive isotopes, phosphorescent chemicals,chemiluminescent chemicals, fluorescent chemicals, enzymes, fluorescentpolypeptides, and epitope tags. The detectable moiety can be a member ofa binding pair, which is identifiable via its interaction with anadditional member of the binding pair, and a label which is directlyvisualized.

When fluorescence labels are used, currently available microscopy andfluorescence-activated cell sorter analysis (FACS) or combination ofboth methods procedures known in the art may be utilized to identify andto quantitate the specific binding members. Anti-TTR antibodies orantigen-binding fragments thereof described herein can carry afluorescence label. Exemplary fluorescence labels include, for example,reactive and conjugated probes, e.g., Aminocoumarin, Fluorescein andTexas red, Alexa Fluor dyes, Cy dyes and DyLight dyes. An anti-TTRantibody can carry a radioactive label, such as the isotopes ³H, ¹⁴C,³²P, ³⁵S, ³⁶Cl, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁶⁷Cu, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹¹⁷Lu,¹²¹I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁹⁸Au, ²¹¹At, ²¹³Bi, ²²⁵Ac and ¹⁸⁶Re. Whenradioactive labels are used, currently available counting proceduresknown in the art may be utilized to identify and quantitate the specificbinding of anti-TTR antibody or antigen-binding fragment to TTR (e.g.,human TTR). In the instance where the label is an enzyme, detection maybe accomplished by any of the presently utilized colorimetric,spectrophotometric, fluorospectrophotometric, amperometric, orgasometric techniques as known in the art. This can be achieved bycontacting a sample or a control sample with an anti-TTR antibody orantigen-binding fragment thereof under conditions that allow for theformation of a complex between the antibody or antigen-binding fragmentthereof and TTR. Any complexes formed between the antibody orantigen-binding fragment thereof and TTR are detected and compared inthe sample and the control. The antibodies or antigen-binding fragmentsthereof described herein can also be used to purify TTR viaimmunoaffinity purification (see e.g., Example 3).

Also included herein is an assay system which may be prepared in theform of a test kit for the quantitative analysis of the extent of thepresence of, for instance, TTR. The system or test kit may comprise alabeled component, e.g., a labeled antibody or antigen-binding fragment,and one or more additional immunochemical reagents. See, e.g., SectionVI below for more on kits.

In some aspects, methods for in vitro detecting TTR in a sample,comprising contacting said sample with an antibody or antigen-bindingfragment thereof, are provided herein. In some aspects, provided hereinis the use of an antibody or antigen-binding fragment thereof providedherein, for in vitro detecting TTR in a sample. In one aspect, providedherein is an antibody or antigen-binding fragment thereof orpharmaceutical composition provided herein for use in the detection ofTTR in a subject or a sample obtained from a subject. In one aspect,provided herein is an antibody or antigen-binding fragment thereof orpharmaceutical composition provided herein for use as a diagnostic. Inone aspect, the antibody comprises a detectable label. In one aspect,TTR is human TTR. In one aspect, the subject is a human.

VI. Kits

Provided herein are kits comprising one or more antibodies orantigen-binding fragments thereof described herein or conjugates (e.g.,detection conjugates) thereof. As provided herein, kits can be used indiagnostic methods. In one aspect, a kit comprises an antibody orantigen-binding fragment thereof described herein, preferably a purifiedantibody or antigen-binding fragment thereof, in one or more containers.

In a specific aspect, kits described herein contain a substantiallyisolated TTR antigen (e.g., human TTR) that can be used as a control. Inspecific aspects, a kit provided herein can include a recombinantlyproduced or chemically synthesized TTR antigen. The TTR antigen providedin the kit can also be attached to a solid support.

In another specific aspect, the kits described herein further comprise acontrol antibody or antigen-binding fragment thereof which does notreact with a TTR antigen. In another specific aspect, kits describedherein contain one or more elements for detecting the binding of anantibody or antigen-binding fragment thereof to a TTR antigen (e.g., theantibody or antigen-binding fragment thereof can be conjugated to adetectable substrate such as a fluorescent compound, an enzyme, anenzymatic substrate, a radioactive compound, or a luminescent compound,or a second antibody or antigen-binding fragment thereof, whichrecognizes the first antibody or antigen-binding fragment thereof, canbe conjugated to a detectable substrate). In a more specific aspect, thedetecting means of the above described kit includes a solid support towhich a TTR antigen is attached. Such a kit can also include anon-attached reporter-labeled anti-mouse/rodent antibody orantigen-binding fragment thereof. In this aspect, binding of theantibody or antigen-binding fragment thereof to the TTR antigen can bedetected by binding of the said reporter-labeled antibody orantigen-binding fragment thereof.

In another specific aspect, the kits described herein further comprise atherapeutic anti-TTR antibody or antigen-binding fragment thereof and/orinformation that a therapeutic anti-TTR antibody or antigen-bindingfragment thereof should be administered when TTR is detected in a sampleusing an anti-TTR antibody or antigen-binding fragment thereof providedherein.

VII. Examples

The examples in this section (i.e., Section VII) are offered by way ofillustration and not by way of limitation.

Materials and Methods Preparation of Fibrillar TTR

Lyophilized purified TTR monomer (1 mg Alexotech, Sweden) was thawed atroom temperature for 10 minutes followed by addition of 0.5 ml ofsterile PBS to a final concentration of 2 mg/ml. TTR was transferred toa new low-binding, sterile 1.5 ml microcentrifuge tube (Protein LoBindTube 1.5, Eppendorf tubes, Cat no.: 022431081) followed by addition of500 μl TTR aggregation buffer ×2 (20 mM sodium acetate (pH 4.3), 200 mMKCl, and 20 mM EDTA) to generate fibrillar TTR in a final volume of 1 mland at a final concentration of 1 mg/ml. The tube was placed in a 37° C.incubation for 72 hours (maximum 4 days). The solution was divided intoaliquots (20-30 μl) into sterile microcentrifuge tubes and stored at−80° C. before use.

Cell Viability Assays

The toxicity of TTR fibrils was assessed by the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide MTT cellviability assay on differentiated rat cardiomyocytes H9c2 cells. For theMTT assay, cells were plated at a density of 1,600 cells per well on96-well plates in 100 μl of culture medium per well. Following 72 hours,the medium was exchanged with 100 μl of fresh medium containing 1% FBSand 10 μM of retanoic acid (RA) for cardiac cell differentiation. After7 additional days, 2.5 μM of TTR fibrils were incubated with CGX304 orsham 1G4-2 for an additional 4 hours at room temperature and added tothe differentiated cardiomyocytes cells for 24 hours at 37° C. Controlsamples were prepared with the addition of identical volumes of buffer.After 24 hours of incubation, the cells were incubated for another 4hours with 100 μl of serum-free Dulbecco's modified Eagle's mediumwithout phenol red, containing 0.5 mg/ml MTT. Then, 100 μl of cell lysissolution (20% SDS, 50% N,N-dimethylformamide) was added to each well,and the samples were incubated overnight at 37° C. to allow completelysis. The absorbance of the formazan was measured at 570 nm in a Tecaninfinite 200 pro microplate reader (Tecan, Männedorf, Switzerland).

Uptake of TTR Fibrils in Mouse Microglia Cells

BV-2 (mouse microglia, ICLC ATL03001) cells were split into 12-wellculture plates (Greiner CELLSTAR multiwell culture plates) on the daybefore the experiment. 0.3 μM TTR fibrils conjugated with Alexa 488,pre-incubated with 0.05, 0.1 0.2, 0.5, 1, 2, 3,or 4 μg/ml CGX304 or 0.1,1, 3, 4 or μg/ml 1G4-2 (sham) for 2 hours at room temperature, wereadded to cells with culture media (RPMI 1640, 10% FBS, 2 mM L-Glutamine,1% Pen/Strep). The conditioned medium was also pre-incubated with CGX304(without fibrillar TTR) for 2 hours as a control. Cells were thenincubated at 37° C. for 24 hours and harvested. Extracellular and cellsurface aggregated TTR conjugated with Alexa 488 in cell pellet wasquenched by incubation of 0.4% trypan blue in PBS (pH 4.4) for 1 minute.Flow cytometry (FL1—blue laser (488 nm) was used to measure TTR fibrils(conjugated to ALEXA fluor 488) uptake indicated as relative geomeanfluorescence intensity (gMFI). Cytochalasin D (Cyto D) is an antibioticwhich is used to inhibit cellular uptake (control).

Uptake of TTR Fibrils in Human Monocytes

THP-1 (human monocytes, ATCC® TIB-202) cells were split into 12-wellculture plates (Greiner CELLSTAR multiwell culture plates) on the daybefore the experiment. 0.3 μM TTR fibrils conjugated with Alexa 488,pre-incubated with either 0.1, or 1 μg/ml CGX304 or 0.1 or 1 μg/ml 1G4-2(sham) for 2 hours at room temperature, were added to cells with culturemedia (RPMI 1640, 10% FBS, 2 mM L-Glutamine, 1% Pen/Strep). Theconditioned medium was also pre-incubated with CGX304 (without fibrillarTTR) for 2 hours as a control. Cells were then incubated at 37° C. for1.5 hours and harvested. Extracellular and cell surface aggregated TTRconjugated with Alexa 488 in cell pellet was quenched by incubation of0.4% trypan blue in PBS (pH 4.4) for 1 minute. Flow cytometry (FL1—bluelaser (488 nm)) was used to measure TTR fibrils (conjugated to ALEXAfluor 488) uptake indicated as relative geomean fluorescence intensity(gMFI). Cytochalasin D (Cyto D) is an antibiotic which is used toinhibit cellular uptake (control).

Fibrillar TTR Protein Levels Post Cell Uptake of Human Monocytes U937

U937 (human monocytes, ATCC® CRL-1593.2) cells were split into 24-wellculture plates (Greiner CELLSTAR multiwell culture plates) on the daybefore the experiment. 0.2 μM TTR fibrils pre-incubated with 0.5 μg/mlCGX304, Ab-D, or 1G4-2 (sham) for 30 minutes at room temperature, wereadded to cells with culture media (RPMI 1640, 10% FBS, 2 mM L-Glutamine,1% Pen/Strep, 10% HEPES and 0.05 mM of 2-mercaptoethanol). Theconditioned medium was also pre-incubated with CGX304 (without fibrillarTTR) for 30 minutes as a control. Cells were then incubated at 37° C.for 0 and 60 minutes and harvested. Harvested cells were washed ×2 withPBS and resuspended in lysis buffer for detecting TTR protein levelsusing western blot. 20 μg of U937 lysates were loaded on a SDS-PAGE (10%acrylamide) and transferred to protran nitrocellulose membranes.Membranes were analyzed by immunoblot for transthyretin using mouseanti-human TTR (mouse IgG1 anti-human transthyretin 39-44, Alexotech;1:1000) followed by peroxidase-AffiniPure goat anti-mouse IgG (Jackson;1:10,000) before enhanced chemiluminescence development. Image Labsoftware (bio-rad) was used to calculate the volume intensity (pixels)normalized to GAPDH for TTR uptake quantification.

Immunopercipitation of Serum-Derived Transthyretin by CGX304

Human sera obtained from TTR amyloidosis patients wereimmunoprecipitated with different amounts of CGX304 (5, 10, and 50 μg)bound to Dynabeads protein A (Dynabeads® Protein A for IP 1 ml, Lifetechnologies, Grand Island, N.Y., USA) following manufacturer'sinstructions, then separated by SDS-PAGE (Bis-Tris 10% acrylamide) andtransferred to protran nitrocellulose membranes. Membranes were analyzedby immunoblot for transthyretin using mouse anti-human TTR (mouse IgG1anti-human transthyretin 39-44, Alexotech; 1:1000) followingperoxidase-AffiniPure goat anti-mouse IgG (Jackson; 1:10,000) beforeenhanced chemiluminescence development. 100 ng of recombinant humanwild-type TTR and PBS were immunoprecipitated with 50 μg of CGX304 boundto Dynabeads as positive and negative control respectively.

Animals

WT rats (Sprague-Dawley) were purchased from Envigo RMS (Israel), Ltd.All housing, breeding, and procedures were performed according to theNational Institute of Health (NIH) Guide for the Care and Use ofExperimental Animals and approved by the Kaplan Medical CenterInstitutional Animal Care and Use Committee.

CGX304 Clears Aggregated TTR Detected by PYP Tech Scan Combined withComputer Tomography Scanning (CT) in the Heart of Rodents

14 male (3 groups) Sprague-Dawley rats (200 grams) were anesthetized byIsoflurane before their chests were opened for TTR fibril injection tothe heart. Matrigel was added to the fibrillar TTR (30 μg in PBS; 50 μlvolume) in a 1:1 volume ratio and loaded (100 μl) to a 1 ml syringeusing a 27G needle and kept on ice until rat was ready for injection.Control animals received sterile PBS and Matrigel (1:1) with no TTR. Asingle needle insertion into the apex of the rat's heart was used tospread the fibrillar TTR for Technetium pyrophosphate (^(99m)Tc-PYP)scintigraphy detection assay model. 4 mCi of ^(99m)Tc-PYP wasadministered intravenously (IV) immediately after surgery. 30 minutesbefore surgery, animals received (IP) CGX304 (5 mg/kg), 1G4-2 (sham), ormatrigel only (negative control) treatment. 1 hour from surgery, animalsunderwent serial ^(99m)Tc-PYP planar cardiac imaging and CT-PET scanningfor transthyretin detection. Cardiac retention was assessedquantitatively (region of interest drawn over the heart, copied, andmirrored over the contralateral chest wall) to calculate aheart-to-contralateral chest wall (Cardiac/Chest wall) ratio. Forbiochemical studies, the heart was removed, tissues were homogenized forprotein purification and CGX304 (mouse IgG1) protein levels weredetected using a calibrated ELISA assay for mouse IgG1 (IgG1 Mouse ELISAKit; ThermoFisher Scientific—cat #88-50410-22).

CGX304 Clears Aggregated TTR Detected by PYP Tech Scan Combined withComputer Tomography Scanning (CT) in Rodent Kidneys

3 Male Sprague-Dawley rats (200 grams) were anesthetized by Isofluranebefore surgery of TTR fibril injection to the kidneys. Aggregated TTR(10m) was mixed together with CGX304 or sham treatment before matrigelwas added in a 1:1 volume ratio and loaded to a 10 μl syringe using a33G needle and kept on ice until rat was ready for injection. Rightkidney received sterile sham treatment, while left kidney receivedCGX304 treatment. A single needle insertion into the left (CGX304) andright (sham) kidneys of rats was used to spread the fibrillarTTR-treatment mix for Technetium pyrophosphate (^(99m)Tc-PYP)scintigraphy detection assay model. 2 mCi of ^(99m)Tc-PYP wasadministered IV immediately after surgery. 1 hour from surgery, animalsunderwent serial ^(99m)Tc-PYP planar cardiac imaging and CT-PET scanningfor transthyretin detection. Kidney retention was assessedquantitatively to calculate a right-to-left kidney (Right/Left kidney)ratio. For biochemical studies, kidneys were removed and tissues wereimmediately frozen and stored at −80° C. until used.

Western Blot for Detection of Aggregated TTR in Rodent Kidneys

12 μg of Left/Right Kidney Protein Lysate were Loaded on a SDS-PAGE (10%Acrylamide) and Transferred to Protran Nitrocellulose Membranes.Membranes were analyzed by immunoblot for transthyretin using rabbitanti-human TTR (mouse IgG1 anti human transthyretin 39-44, Alexotech;1:1000) followed by peroxidase-AffiniPure goat anti-rabbit IgG (Jackson;1:10,000) before enhanced chemiluminescence development.

CGX304 Pharmacokinetics/Pharmacodynamics—Antibody Clearance/RatPrealbumin Levels in Serum

Rats were divided into 3 groups of n=3 to characterize the plasmaconcentration-endogenous TTR effect relationship for CGX304 in apharmacokinetics/pharmacodynamics model. Each group received a differentinitial concentration of CGX304 (0.5 mg/kg, 2.5 mg/kg, or 10 mg/kg) IV.Blood samples were collected 1 hour, 2 hours, 72 hours, 7 days, and 28days post CGX304 IV injection. For biochemical studies, the heart wasremoved 28 days post IV injections, and tissues were homogenized forprotein purification. CGX304 (mouse IgG1) protein levels (PK) in bloodserum and heart tissue total protein content lysate were detected usinga calibrated ELISA assay for mouse IgG1 (IgG1 Mouse ELISA Kit;ThermoFisher Scientific—cat #88-50410-22), while free endogenous ratpre-albumin (TTR) levels were detected using a calibrated ELISA assayfor rat TTR (Rat TTR/Transthyretin ELISA Kit (Sandwich ELISA)—LS-F9963).

Immunohistochemistry

Human heart tissue from TTR amyloidosis patients were post-fixed in 4%formalin for 24 hours and embedded in paraffin. Hearts were cut togenerate 8-16 μm thick sections. A series of sections was stained foreither CGX304 or congo red (abcam, ab150663—manufacturer protocol).Tissue was washed for 5 minutes ×3 times in xylene followed by ×2 in100% ethanol. Than tissue was incubated in 0.3% H₂O₂ (in methanol) for15 minutes in the dark, rinsed in DDW for 1 minute, followed by antigenretrieval (citrate buffer pH 6 xl in pressure chamber—120° C. for 30minutes followed by 20 minutes of cooling), followed by CGX304 (1:1000)antibody incubation for 1 hour at room temperature. Then, sections wereincubated in secondary antisera against mouse IgG (DAKO Envision FLEXSM802, manufacturer dilution). Antibody labeling was visualized byexposure to 0.5 mg/ml 3,3′ diaminobenzidine (DAB—Zymotec), 2.5 mg/mlnickel ammonium sulfate, and 0.03% H₂O₂ in Tris buffer followed byhematoxylin (Mayer's—abcam—ab220365) counter stain. Sections weremounted on subbed slides, dehydrated to xylene and coverslipped withxylene base mounting buffer.

Generation of Short Term Rat Model of wt ATTR and In Vivo Testing ofCGX304

20 male (4 groups) Sprague-Dawley rats (200 grams) were anesthetized byIsoflurane before their chests were opened for aggregated TTR injectionto the heart. Matrigel was added to aggregated TTR (30 μg in PBS; 50 μlvolume) in a 1:1 volume ratio and loaded (100 μl) to a 1 ml syringeusing a 27G needle and kept on ice until the rat was ready forinjection. Control animals received sterile PBS and Matrigel (1:1) withno TTR. A single needle insertion into the apex of the rat's heart wasused to spread the aggregated TTR for Technetium pyrophosphate(^(99m)Tc-PYP) scintigraphy detection assay model. Four mCi of ^(99m)Tc-PYP were administered intravenously (IV) immediately after surgery.30 minutes before surgery, animals received (IP) CGX304 (5 or 10 mg/kg)or non-relevant antibody 1G4-2 (sham) treatment. One hour later, animalsunderwent serial ^(99m)Tc-PYP planar cardiac imaging and SPECT/CTscanning for TTR detection. Cardiac retention was assessedquantitatively using a volume of interest (VOI) over the left ventricle.Background was assessed using a posterior chest wall VOL For biochemicalstudies, hearts were removed and tissues were immediately frozen andstored at −80° C. until used.

Immunohistochemistry of Aggregated TTR in Apex of Injected Rats

Rat hearts were post-fixed in 4% formalin for at least 4 hours andembedded in paraffin. Hearts were cut to generate 4 μm thick sections. Aseries of sections were stained with murine CGX304 or control isotypeantibody. Tissue was washed for 5 minutes 3 times in xylene followed by2 times in 100% ethanol. Then tissue was incubated in 0.3% H₂O₂ (inmethanol) for 15 minutes in the dark, rinsed in DDW for 1 minute,followed by antigen retrieval (citrate buffer pH 6 xl in pressurechamber—120° C. for 30 minutes followed by 20 minutes of cooling),followed by murine CGX304 (1:1000) antibody incubation for 1 hour atroom temperature. Then, sections were incubated in secondary antiseraagainst mouse IgG (DAKO Envision FLEX SM802, manufacturer dilution).Antibody labeling was visualized by exposure to 0.5 mg/ml 3,3′diaminobenzidine (DAB—Zymotec), 2.5 mg/ml nickel ammonium sulfate, and0.03% H₂O₂ in Tris buffer followed by hematoxylin(Mayer's—abcam—ab220365) counter stain. Sections were mounted on subbedslides, dehydrated to xylene, and coverslipped with xylene basedmounting buffer.

Echocardiography in Rat Short Term Rat Model of wtATTR

Echocardiography was performed using a 12 MHz transducer (VIVID 6, GEmedical) Conventional Left ventricular (LV) structure, and function wasassessed from the parasternal, long (LAX) and short (SAX) axis views (atthe level of the papillary muscles). Mitral inflow and aortic outflowwere recorded by Doppler. Measurements were obtained off-line (EchoPACBT13, General Electric). Chamber diameters, wall-thickness, andfractional shortening (FS) were measured by M-mode, in the SAX view,according to the “leading edge to leading edge” convention.Two-dimensional speckle-tracking strain analysis was performed offline.The LV was divided into 6 segments at the parasternal short axis view ofthe mid left ventricle as defined by the American Society ofEchocardiography. Parameters including the peak systolic radial strainand radial strain rate (RS and RSr) and peak systolic circumferentialstrain and circumferential strain rate (SC and CSr) of each of the 6segments were measured.

Immunohistochemistry of wtATTR Human Cardiac Biopsies

Human heart tissues from wtTTR amyloidosis patients were post-fixed in4% formalin for 24 hours and embedded in paraffin. Hearts were cut togenerate 8-16 μm thick sections. A series of sections was stained witheither mouse CGX304, control mouse IgG, or Congo red (abcam,ab150663—manufacturer protocol). Tissue was washed for 5 minutes 3 timesin xylene followed by 2 times in 100% ethanol. Then, tissue wasincubated in 0.3% hydrogen peroxide (H₂O₂) (in methanol) for 15 minutesin the dark, rinsed in DDW for 1 minute, followed by antigen retrieval(citrate buffer pH 6 1 time in pressure chamber—120° C. for 30 minutesfollowed by 20 minutes of cooling), followed by CGX304/control IgG(1:1000) antibody incubation for 1 hour at room temperature. Then,sections were incubated in secondary antisera against mouse IgG (DAKOEnvision FLEX SM802, manufacturer dilution). Antibody labeling wasvisualized by exposure to 0.5 mg/ml 3,3′ diaminobenzidine (DAB—Zymotec),2.5 mg/ml nickel ammonium sulfate, and 0.03% H₂O₂ in Tris bufferfollowed by hematoxylin (Mayer's—abcam—ab220365) counter stain. Sectionswere mounted on subbed slides, dehydrated to xylene, and coverslippedwith xylene based mounting buffer.

Generation of an ELISA to Test the Presence of Circulating AggregatedTTR Employing CGX304 Derivatives

Aiming to validate the ability of CGX304 to detect circulating levels ofaggregated TTR, a capture ELISA was established. The details of thepatients with wtATTR cardiac amyloidosis and control heart failure (HF)patients are provided in Table

TABLE 3 Clinical characteristics of patients with wtATTR versus HFpatients wtATTR HF controls (n = 27) (n = 26) P Age; years [+/−SD] 70.2[+/−10.6] 67.4 [+/−12.8] 0.382 Male Sex; % [n] 51.9 [14] 69.2 [18] 0.264NYHA I-II; % [n] 37.0 [10] 69.2 [18] 0.028 NYHA III-IV; % [n] 63.0 [17]30.8 [8] 0.028 Hypertension; % [n] 81.5 [22] 88.5 [23] 0.704 Diabetes: %[n] 48.1 [13] 34.6 [9] 0.406 Obesity; % [n] 48.1 [13] 34.6 [9] 0.406Obstructive coronary 33.3 [9] 26.9 [7] 0.766 artery disease; % [n]Atrial fibrillation; % 44.4 [12] 53.8 [14] 0.587 [n] Pacemaker/CRT/ICD;66.7 [18] 34.6 [9] 0.029 % [n] Pacemaker; % [n] 29.6 [8] 0 [0] 0.004 GFRwith eGFR 40.7 [11] 30.8 [8] 0.569 <60; % [n] ACEi/ARB/ARNI; % 88.9 [24]96.2 [25] 0.610 [n] Beta blockers; % [n] 88.9 [24] 96.2 [25] 0.610 MRA;% [n] 44.4 [12] 46.2 [12] 1.000 Diuretics; % [n] 77.8 [21] 61.5 [16]0.241 LVEF; % [+/−SD] 38.7 [+/−10.7] 40.4. [+/−13.1] 0.620

ELISA plates were coated with 100 μl/well of 4 μg/ml NeutrAvidin(ThermoFisher Scientific—cat #31000) and incubated overnight at 4° C.Wells were aspirated and washed 3 times (300 μl/well) with washingbuffer (0.1% Tris Buffered Saline with 0.1% Tween 20 (TBST)) followed by1 hour incubation at room temperature with 300 μl/well blocking buffer(5% BSA/TBST). Plates were then washed 3 times and incubated for 1.5hours at room temperature with 100 μl/well biotinylated mouse CGX304 (2μg/ml). Plates were washed three times, standard TTR and human serumsamples were diluted in sample buffer (0.1% BSA/PBS), loaded into theplates (100 μl/well), and incubated for an additional 1.5 hours at roomtemperature. After additional washing steps, HRP-labeled mouse CGX304was added to the wells (100 μl/well) and incubated for 1 hour at roomtemperature. Development was performed with substrate reagent containingtetramethylbenzidine and hydrogen peroxide (R&D systems, cat # DY999).The TTR concentration in samples was calculated from a standard curveranging from 3.9 to 2000 ng/ml. The experiment was validated three timeswith triplicate sample repetition.

Statistical Analysis

Values shown in the Figures are presented as mean+/−SEM. P values fordetermination of the statistical significance of differences werecalculated by means of paired, two-tailed Student's t test, one-wayANOVA with a post hoc Dunnet's or one-way ANOVA with Tukey's post test.The reproducibility of RS and CR measurements was analyzed with repeatedmeasurements, in a blinded fashion, by an observer at two different timepoints in 5 randomly selected studies. Intra-observer agreement wasevaluated by Bland-Altman analysis. In order to decrease non-constantvariability, logarithmic transformation was performed. Intra-classCorrelation Coefficient was calculated.

Example 1: Generation of Monoclonal Antibodies

Several clones of monoclonal antibodies (mAbs) were produced accordingto standard protocols by Balb/C mice immunization with 50 μg of variantmixtures of transthyretin (TTR) (Alexotech, Sweden) followed by threeadditional boosts. After confirming the presence of polyclonal anti-TTRantibodies (Abs) in the sera, mice were sacrificed. Cells were isolatedfrom their spleens and hybridized with an SP2/0 myeloma line, followedby clonal screening for binding to TTR. The hybridomas were then grownin serum-free media for 2-3 weeks, and media were collected andconcentrated by 30 kDa centricons (Biological Industries, Israel).Cross-reactivity of mAbs with human TTR was confirmed by ELISA.

A standard ELISA was performed using variant mixtures of human TTR asthe coating protein. As shown in FIGS. 1A and 1B, clones Ab-B, CGX304,Ab-C, Ab-D, Ab-E and Ab-F bound to the coating protein in aconcentration-dependent manner. Clone Ab-A showed lower affinity bindingunder these assay conditions. Sham did not show binding at all (FIG.1B).

Example 2: High Affinity Binding of Unique Clones

Two selected unique clones (CGX304 and Ab-D) were chosen from a panel ofa mixture of wild-type and mutated TTR-binding mouse IgG by their highaffinity binding to monomer and fibrillar TTR seen in ELISA assay (FIG.2). 1G4-2 was used as mouse IgG sham control. CGX304 clone was selectedand tested for its high affinity to TTR fibril and monomer forms insurface Plasmon resonance (Biacore SPR system, GE Healthcare LifeSciences) (FIG. 3).

Example 3: CGX304 Immunoprecipitates Transthyretin from TTR AmyloidosisPatients' Sera

Human serum derived from transthyretin amyloidosis patients wereimmunoprecipitated with CGX304 to determine the antibody's ability tobind endogenous human transthyretin. Three different amounts of CGX304(50 μg, 10 μg, and 5 μg) were able to immunoprecipitate TTR fromtransthyretin amyloidosis patients' sera (FIG. 4). Samples were stainedwith a commercial polyclonal rabbit anti-human TTR antibody (GTX33557,GeneTex) (FIG. 4). Human recombinant TTR immunoprecipitation with 50CGX304 was performed as positive control.

Example 4: CGX304 Avidly Binds Heart Tissues from Patients with TTRAmyloidosis

CGX304 was able to bind avidly to its target transthyretin in a numberof human heart transthyretin amyloidosis tissue samples seen byimmunohistochemistry staining (FIG. 5), demonstrating its ability torecognize modified forms of TTR in multiple patients with TTRamyloidosis.

Example 5: CGX304 Protects Rat Cardiomyocytes Cells from Fibrillar TTRInduced Toxicity

The cytotoxicity of TTR fibrils was assessed on differentiated ratcardiomyocytes H9c2 cells using the3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay(FIG. 6). Fibrillar TTR had a toxic effect on cells in lowconcentrations as seen by the cell viability assay (FIG. 6). Incubationof fibrillar TTR in the presence of increasing concentrations of CGX304,showed an increase in cell viability in a dose-dependent manner, whileno affect was seen in the presence of sham (1G4-2) (FIG. 6). Incubationof TTR fibrils in the presence of increasing concentrations of CGX304showed a high increase in cell viability at concentrations of 8 μg/ml(˜61%), 6 μg/ml (˜43%), and 4 μg/ml (˜33%) CGX304. CGX304 concentrationsof 2 μg/ml and 1 μg/ml almost had no effect on cell viability (FIG. 6).

Example 6: CGX304 Increases Uptake of TTR Fibrils in Human Monocytes andMouse Microglia

One of the main functions of macrophages and microglia in the immunesystem is phagocytosis. Phagocytosis is a major mechanism used to removepathogens and cell debris, and it helps macrophages and microglia serveas antigen presenting cells, which are essential for T-cell immuneresponse triggering. Flow cytometry was used to measure TTR fibrilsconjugated to Alexafluor 488 cellular uptake (calculated by relativegeomean fluorescent intensity (gMFI)) (FIGS. 7A, 7B, 7C, and 7D). Flowcytometry analysis shows that CGX304 increases mouse and humanmacrophages cellular uptake of fibrillar TTR conjugated to Alexafluor488 (CGX304—relative gMFI—101) in a dose dependent manner compared tocells with no treatment (PBS—relative gMFI—58) and sham (1G4-2—relativegMFI—51) as seen in FIGS. 7A and 7B. FIGS. 7C and 7D show that bothCGX304 (FIG. 7D—volume intensity ˜0.9) and Ab-D (FIG. 7D—volumeintensity ˜1) increase human monocytes (U937) cellular uptake offibrillar TTR after 60 minutes of incubation (FIGS. 7C and 7D) comparedto sham (1G4-2) (FIG. 7D—volume intensity ˜0.6). No significantdifference was seen after zero (0) minutes (cell lysate is preparedimmediately after addition of CGX304/Ab-D/1G4-2—TTR complex) ofincubation with cells (FIGS. 7C and 7D).

Example 7: CGX304 Clears Aggregated TTR Detected by PYP Scans Combinedwith Computer CT in Rodent Hearts

Technetium pyrophosphate (99mTc-PYP) imaging has been used to diagnosetransthyretin cardiac amyloidosis (ATTR-CA). 99mTc-PYP is FDA approved(for bone imaging as an adjunct in the diagnosis of acute myocardialinfarction or as blood pool imaging agent), is readily available, andhas been the preferred radiopharmaceutical for assessment of cardiacamyloidosis. The mechanism of myocardial transthyretin amyloid uptake of99mTc-PYP is unclear, but it has been suggested that calcium in amyloiddeposits binds to phosphate in these radiotracers.

Here, a new rat model was used to detect cardiac deposition of thewild-type, aggregated, misfolded TTR protein using 99mTc-PYP imaging(FIGS. 8A, 8B, and 8C). The advantage of this method is the ability totest and monitor the effects of new drugs on aggregated TTR clearance ina short period of time. FIG. 8C shows that CGX304 was detectable in therat heart after treatment. PET-CT images show TTR clearance in the heartof CGX304 treated animals compare to sham (FIG. 8A; white arrows). FIG.8B shows elevated levels of fluorescence intensity (F.I.) in sham+TTRtreatment group (˜2.3) compared to CGX304+TTR treatment group of animals(˜1.5). There was no significant difference between the animal groupwith CGX304 treatment (CGX304+TTR) compared to the control group thatreceived sham treatment and no TTR (sham-TTR; ˜1.3). This resultdemonstrates the CGX304 clearance effect on aggregated TTR in the heartof rats.

Kidney impairment and proteinuria are also clinical features of ATTR.Nephropathy affects patients with late-onset neuropathy, low penetrancein the family, and cardiac dysrhythmias. Amyloid renal deposits commonlyoccur, even in the absence of urinary abnormalities. Therefore,technetium pyrophosphate (99mTc-PYP) imaging was also used for detectionof kidney deposition of the wild-type aggregated misfolded TTR protein(FIGS. 9A, 9B, and 9C). The advantage of this method is the in vivocomparison between two identical organs, e.g., the ability to test theeffects of a drug by comparison of the two sides (treated anduntreated). PET-CT images show TTR clearance in the CGX304-treated leftkidney compared to the sham-treated right kidney (FIG. 9A; whitearrows). FIG. 9B shows elevated levels of fluorescence intensity (F.I.)in the right kidney as compared to the left kidney (right/leftkidney >1) in all animals, indicating there was higher levels of TTR inthe right kidney (sham) compared to the left kidney (CGX304). Theimmunoblot image in FIG. 9C demonstrates the effect of CGX304 on TTRclearance. There were higher tracers of TTR protein in the right kidney(R) compare to the left (L) in all animals.

Example 8: CGX304 Pharmacokinetics and Pharmacodynamics

FIG. 10A shows the PK values (mouse IgG1 concentration (ng/ml)) in ratserum 1 hour, 24 hours, 72 hours, 7 days, and 28 days post intravenous(IV) injection. Each group received a different initial concentration ofCGX304 (0.5 mg/kg, 2.5 mg/kg, and 10 mg/kg) IV. CGX304 half-life valueis 187.5 hours (FIG. 10A). FIG. 10B shows free endogenous ratpre-albumin (TTR) levels (PD values) in blood serum of same group ofanimals. FIG. 10C shows levels of CGX304 in rat hearts 28 days after IVadministration at various doses.

Example 9: CGX304 Stains Aggregated TTR in Cardiac Samples from Patientswith wtATTR

To test the ability of mouse CGX304 to detect aggregated TTR inpatients, tissue samples from hearts of human WT ATTR cardiacamyloidosis patients were immunostained with mouse CGX304 or control IgGand compared to Congo red staining. CGX304 was able to bind avidly toits target transthyretin in a number of human heart transthyretinamyloidosis tissue samples as seen by immunohistochemistry staining(FIG. 12). These results demonstrate the ability of CGX304 to recognizemodified forms of TTR in multiple patients with significantly highersensitivity than congo red staining. There was no staining of modifiedforms seen using the mouse control IgG antibody.

Example 10: CGX304 Clears Aggregated TTR Evident by In Vivo PYPScintigraphy Signals

A short term rat model of wtATTR was used to assess the ability ofCGX304 to clear aggregated TTR. Aggregated TTR was injected into thehearts of rats that received either CGX304 or a control (sham) antibody,and PYP was used to detect aggregated TTR (as described in the materialsand methods).

The results are shown in FIG. 13. Detectable levels of aggregated TTRwere seen in the rat heart after treatment. FIG. 13 shows a decrease inPYP cardiac uptake (% Normalized TTR uptake ratio) in 10 mg/kg(p=0.0079) and 5 mg/kg (p=0.042) CGX304+TTR treatment groups (70.87% and73.95%, respectively) relative to the sham+TTR treatment group (100%).There was not a significant difference between the group treated with 1mg/kg CGX304 (CGX304+TTR) compared to the control group that receivedsham treatment and TTR (sham+TTR; 94.39%). This result demonstrates thatCGX304 clears and degrades the aggregated TTR in rat hearts.

Example 11: CGX304 Stains Aggregated TTR in Apex Injected Rats

The ability of CGX304 to detect aggregated TTR was also observed in ratswith aggregated TTR injected into the heart apex. CGX304 was able tobind avidly to aggregated TTR injected into the apex of rats hearts asseen by immunohistochemistry staining (FIG. 11). These resultsdemonstrate the ability of CGX304 to recognize the injected modified TTRin the PYP rat model. There was no staining of TTR seen in control rats(rats injected with sham (—TTR)).

Example 12: Functional Improvement in Cardiac Performance by CGX304 inthe Experimental ATTR Model

Echocardiography was performed pre and post aggregated TTR injections(with/without IV treatment) in the short term rat model of wtATTR. Ratswere injected with aggregated TTR injection to the hearts followed byCGX304 (5 mg/kg) or sham treatment. The resulting changes in leftventricular size and function are shown in Table 4.

TABLE 4 Echocardiographic characteristics of rats before and after TTRinjection with and without CGX304 Sham (n = 5) CGX304 (n = 4) Std. Std.Mean Deviation P value Mean Deviation P value RS (%) Baseline 33.20 7.460.02 30.25 5.32 0.051 post 23.40 4.51 27.00 6.27 RSr-S (s-1) Baseline6.20 2.39 0.90 8.39 2.87 0.740 post 6.00 1.87 7.75 1.26 RSr-E (s-1)Baseline −7.50 3.91 0.27 −6.00 2.16 0.916 post −5.20 0.84 −6.25 2.99RSr-A (s-1) Baseline −5.36 1.32 0.20 −4.67 3.06 1.000 post −2.16 4.07−4.67 0.58 CS (%) Baseline −10.80 2.77 0.42 −10.75 2.99 0.448 post−11.98 1.45 −12.00 2.16 CSr-s (s-1) Baseline −5.00 0.71 0.01 −4.13 0.630.080 post −3.86 0.77 −4.75 0.96 CSr-E (s-1) Baseline 3.20 0.45 0.203.58 0.72 0.336 post 4.06 0.90 4.50 1.29 CSr-A (s-1) Baseline 3.60 1.140.07 2.75 0.50 0.06 post 3.00 1.00 3.50 0.58 CSr-E/CSr-A Baseline 0.980.40 0.006 1.29 0.40 0.78 post 1.42 0.30 1.27 0.20 DD (mm) Baseline 0.660.09 0.62 0.65 0.10 0.18 post 0.68 0.08 0.70 0.08 SD (mm) Baseline 0.400.07 0.62 0.35 0.06 0.39 post 0.42 0.04 13.83 26.78 FS (%) Baseline38.00 4.95 0.68 45.75 5.56 0.14 post 36.60 2.30 38.00 3.16 ME/MABaseline 1.76 1.05 0.55 1.31 0.25 0.89 post 1.36 0.59 1.27 0.45 E/E′Baseline 0.23 0.07 0.38 0.15 0.01 0.67 post 0.15 0.05 0.15 0.01 TDI-IEBaseline 3.33 0.58 0.53 4.50 0.71 0.67 (cm.s) post 4.00 1.00 3.50 0.71RS = Radial strain, RSr = Radial strain rate, CS = Circumferentialstrain, CSr = Circumferential strain rate, DD diastolic diameter, SD =systolic diameter, FS = fractional shortening, ME-mitral E wave, MA =mitral A wave, TDI tissue Doppler imaging. All the P values arecomparison of baseline and post injection image.

There was no change in left ventricular dimension between the groups.There was also no change in the conventional systolic functionparameters, e.g., fractional shortening. However, in 5 control ratsthere was a significant decrease in radial strain (FIGS. 14A and 14D)and in circumferential strain rate S (FIG. 14B) (p=0.017 and p=0.007,respectively). In the CGX304-treated group, these changes in theparameter of systolic deformation were blunted and not statisticallysignificant. There were no significant changes in diastolic function,including the ratios ME/MA and E/E′. The circumferential strain rate Eincreased, and the A decreased not significantly in both groups. Theratio of circumferential strain rate E to A changed less in thetreatment group (FIG. 14C). The functional changes seen in deformationanalysis are usually more sensitive and appear earlier than the changein LVEF and this was shown in various experimental and clinical studies(Popovic et al., AM J. Physiol Heart Circ Physiol 292(6): H2809-16(2007); Pagourelias et al., Circ Cardiovasc Imaging 10(3):e005588(2017)). The reproducibility of RS and CS measurements are shown in FIG.14E. The assessment of RS and CS parameters shows a good intra-observeragreement with low mean bias (0.018±0.208, 95% limits of agreement of−0.3892-0.426) and good reproducibility, with an intra-class correlationcoefficients of 0.974 (95% CI 0.932-0.990).

Example 13: An ELISA Employing CGX304 Detects High Levels of ShedAggregated TTR in the Sera of Patients with wtATTR Cardiac Amyloidosis

An ELISA assay employing Ab-A detected the presence of circulatingaggregated TTR in sera of patients with wtATTR cardiac amyloidosis (FIG.15, wtATTR) compared to control heart failure patients (FIG. 15,Control). The assay was conducted on 43 patients with wtATTR cardiacamyloidosis and 42 control patients with heart failure matched by theirbaseline clinical characteristics. The diagnosis of wtATTR cardiacamyloidosis was based on PYP scintigraphy after ruling out light chaincardiac amyloidosis.

1. An antibody or antigen-binding fragment thereof capable of binding human transthyretin (TTR), wherein the antibody or antigen-binding fragment thereof comprises a complementary determining region (CDR) H1 comprising the amino acid sequence set forth in SEQ ID NO: (GYTFTSYY), a CDR H2 comprising the amino acid sequence set forth in SEQ ID NO:2 (IYPGNVNT), a CDR H3 comprising the amino acid sequence set forth in SEQ ID NO:3 (ARTYFDY), a CDR L1 comprising the amino acid sequence set forth in SEQ ID NO:4 (SSVSY), a CDR L2 comprising the amino acid sequence set forth in SEQ ID NO:5 (DTS), and a CDR3 L3 comprising the amino acid sequence set forth in SEQ ID NO:6 (QQWSSKSFT.)
 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises the same amino acid sequence as the VH of the antibody produced by the hybridoma cell line deposited at the ATCC® as deposit number PTA-125005 on Mar. 14, 2018 and wherein the VL comprises the same amino acid sequence as the VL of the antibody produced by the hybridoma of ATCC® deposit number PTA-125005.
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 14. An antibody or antigen-binding fragment thereof that specifically binds to human TTR, wherein the antibody or antigen-binding fragment thereof comprises the CDR H1, CDR H2, CDR H3, CDR L1, CDRL2, and CDR L3 of the antibody produced by the hybridoma of ATCC® deposit number PTA-125005.
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 16. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds to human TTR monomers and/or human TTR fibrils.
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 21. An isolated antibody or antigen-binding fragment thereof that binds to the same epitope of human TTR as the antibody or antigen-binding fragment thereof of claim 1 or competitively inhibits binding of the antibody or antigen-binding fragment thereof of claim 1 to human TTR.
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 26. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment is an antigen-binding fragment, wherein the antigen-binding fragment comprises a Fab, Fab′, F(ab)₂, single chain Fv(scFv), disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH₂, minibody, F(ab′)₃, tetrabody, triabody, diabody, single domain antibody, DVD-Ig, Fcab, mAb², (scFv)₂, or scFv-Fc.
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 29. An isolated polynucleotide comprising a first nucleic acid molecule encoding the heavy chain variable region or heavy chain of the antibody or antigen-binding fragment thereof of claim 1 and a second nucleic acid molecule encoding the light chain variable region or light chain of the antibody or antigen-binding fragment thereof of claim
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 36. An isolated vector comprising the polynucleotide of claim
 29. 37. A host cell comprising the polynucleotide of claim
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 39. A method of producing an antibody or antigen-binding fragment thereof that binds to human TTR comprising culturing the host cell comprising the isolated polynucleotide of claim 29 so that the first nucleic acid molecule and the second nucleic acid molecule are expressed and the antibody or antigen-binding fragment thereof is produced.
 40. An isolated antibody or antigen-binding fragment thereof that is produced by the method of claim
 39. 41. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of claim 1 and a pharmaceutically acceptable excipient.
 42. A method for detecting TTR in a sample or precipitating TTR from a sample comprising contacting the sample with the antibody or antigen-binding fragment thereof of claim
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 45. A method for increasing the uptake of TTR aggregates by an immune cell exposed to TTR aggregates comprising contacting the immune cell with the antibody or antigen-binding fragment of claim
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 48. A method for decreasing the toxicity of TTR aggregates on a cell exposed to TTR aggregates comprising contacting the cell with the antibody or antigen-binding fragment thereof of claim
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 50. A method of inhibiting the accumulation of TTR aggregates in an organ exposed to TTR aggregates comprising contacting the organ with the antibody or antigen-binding fragment thereof of claim
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 55. A method of treating or preventing a TTR amyloidosis in a subject comprising administering to the subject the antibody or antigen-binding fragment thereof of claim
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 61. A method for diagnosing a TTR amyloidosis in a subject comprising administering to the subject the antibody or antigen-binding fragment thereof of claim
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 64. A kit comprising the antibody or antigen-binding fragment thereof of claim 1 and a) a detection reagent, b) TTR antigen, c) a notice that reflects approval for use or sale for human administration, or d) a combination thereof.
 65. A method for testing the activity of a TTR-binding compound comprising administering the TTR-binding compound and TTR fibrils to a rodent.
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